CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/801,112, filed March 15, 2013, the entire contents of which are hereby incorporated by reference in their entirety for ail purposes.

FIELD OF THE INVENTION

The invention relates to a novel composition and polymorphs of sodium 4-((3-(4- eyclohexylpiperazin- 1 -yl)-6-oxo-6H-anthra[ 1 ,9-cd]isoxazol-5-yl)amino)benzate, particularly the polymorphic form A, mixtures of the polymorphs, process for the preparation thereof and the use thereof in a pharmaceutical composition containing thereof.

BACKGROUND

The free acid of compound 4-((3-(4-cyclohexylpiperazin-l-yl)-6-oxo-6H~ant,hra[l,9- cd]isoxazol~5~yi)amino)henzoic acid ("Compound 701") and its pharmaceutical composition are known in US Patent Application No. 201 10301 133 Al (corresponding PCT Patent Application, WO2009/067197, which is hereby incorporated by reference) and has the following chemical structure:

Compound 701 , and the physiologically acceptable salts thereof, have valuable pharmacological properties. Compound 701 is a receptor tyrosine kinase inhibitor, particularly a potent NGF receptor TrkA inhibitor which, by virtue of its pharmacological properties, may be used, for examples, for the treatment and/or prevention of acute and chronic pain, cancer, restenosis, atherosclerosis, psoriasis, thrombosis, skin diseases, inflammation, inflammation related diseases, or a disease, disorder or injury relating to dysmyelination or demyelination. Other possible therapeutic applications can be found in WO2009/067197, the contents of which are hereby referred to.

Due to the limited solubility of the free acid Compound 701 , it is very difficult to achieve practical oral bioavailability in the in- vivo systems, for example, in rat. Salt, formation has been widely used in the art to improve lipophilic, water solubility and/or oral bioavailability, among other issues in the industry. However, it is problematic and huge challenge for selection and testing of the enormous numbers of possible salt form s in practice, since Compound 701 is zwitterionic molecule, there are vast amount of counterions and/or molecules that could form salts or co-crystals with Compound 701 and they can be from either (i) anionic counter-ions, for examples, acetate, benzoate, bicarbonate, bisulfite, norate, bromide, carbonate, chloride, citrate, formate, fumerate, glcuconate, giueuronate, hydrochloride, malate, nitrate, phosphate, salicylate, succinate and tartrate; (ii) cationic counter-ions, for examples, ammonium, piperazine, diethlyamine, diethanolamine, imidazole, diethylammonium., ethylenediamine, betaine, lithium, sodium, potassium, calcium, magnesium, aluminum, zinc, bismuth and stomum; or (iii) z itteriomc molecules, for examples, glycine, bicine, tricine, sulfamic acid, iysergic acid, and psilocybin. Furthermore, each salt or co-crystal forms may form various polymorphs with various degrees of physicochemical properties in terms of, for examples, solubility, stability, and oral bioavailability, it is therefore problematic and huge challenge in practice.

Another aspect which is important in drug development is that the active substance should have the most stable possible crystalline morphology for the pharmaceutical quality of a medicinal formulation, if this is not the case, the morphology of the active substance may change in certain circumstances under the conditions of manufacture of the preparation. Such a change may in turn affect the reproducibility of the manufacturing process and thus lead to final formulations which do not meet the high quality requirements imposed on pharmaceutical formulations. To this extent it should generally be borne in mind that any change to the solid state of a pharmaceutical composition which can improve its physical and chemical stability gives a significant advantage over less stable forms of the same drug. SUMMARY OF THE INVENTION The present invention provides, among other things, small molecule compounds and their salts or solvates in various crystalline forms and amorphous form as NGF receptor TrkA inhibitor and/or antagonist for the preparation of a medicament for the treatment and/or prevention of diseases associated, directly or indirectly with modulation of activity or expression of TrkA protein kinase, which include pain, cancer (e.g., pancreatic cancer, gastric cancer, esophageal cancer, gastrointestinal cancer, colorectal cancer, lung cancer, liver cancer, brain cancer or human neuroblastoma, glioblastoma and medulloblastoma, retinoblastoma, leukemia, lymphoma, melanoma, malignant mesothelioma, breast cancer, bladder cancer, ovarian cancer, prostate cancer, thyroid cancer, squamous cell carcinomas), itching, atopic dermatitis, scabies, pityriasis, inflammatory bowel disease, inflammatory arthritis, asthma, human airway diseases, Chagas' disease, parasitic diseases, Alzheimer's, restenosis, atherosclerosis, psoriasis, skin diseases, thrombosis, or a disease, disorder or injury relating to dysmyelination or dernyehnation or the disease or disorder associated with abnormal activities of TR protein kinases.

In one aspect, the present invention provide a compound of the following structure in various crystalline forms and amorphous form:

Sodium 4-((3-(4~cyclohexyipiperazin~l-yl)~6-oxo-6H-anthra[l ,9-cd]isoxa.zoi-5- y l)amino)benzoate .

In one embodiment, a crystalline polymorph of Compound I is crystalline polymorph Form A. In another embodiment, the crystalline polymorph exhibits an x-ray powder diffraction pattern having peak positions at degree two-theta of about: 10.0±0.3, 2G.1+0.3, and 23.6±0.3. In another embodiment, the crystalline polymorph exhibits an x-ray powder diffraction pattern having peak positions at degree two-theta of one or more of about: 14.5±0.3 and 18.1±0.3, and about: 9.7+0.3 and 21.2±0.3. In a further embodiment, the crystalline polymorph exhibits an x- ray powder diffraction pattern having at least three, at least, five, at least, seven, at least ten, or all, of the following peak positions at degree two-theta of about: 7.160, 8.757, 9.820, 10.161 , 12.459, 14.641, 15.219, 17.680, 18.240, 19.104, 20.220, 21.381 , 22.579, 23.721 , 24.898, 25.761, 25.522, 27.161 , 28.321 , 28.321, 29.481, 30.921 , and 34.281.

In a further embodiment, a crystalline polymorph of Compound I of claim 1 is crystalline polymorph Form. B. In a still further embodiment, the crystalline polymorph exhibits an x-ray powder diffraction pattern having three or more, or five or more, or seven or more, or all of, the peak positions at degree two-theta selected from the group consisting of about: 9,8±0,3,

1Q.2±Q.3, i 4.5 - 0.3. 17.8±0.3, 18.5±0.3, 19.6±0.3, 2.1.0±Q.3, 21 .7 - 0.3. and 23..1 ±0.3. In yet another embodiment, the crystalline polymorph exhibits an x-ray powder diffraction pattern having three or more, or five or more, or seven or more, or ten or more, or all of, the peak positions at degree two-theta selected from the group consisting of about: 9.8±0.3, 10.2±0.3, 14.5±0.3, 1 7.8 - 0.3. 1 8.5 0.3. 19.6±0.3, 21 .0±0.3, 21.7±0.3, 23.H0.3, 25.0±0.3, 25.6±0.3, 28.4 - 03. 29.4 . 0.3. 30.2 · 0.3. and 3 i .6 · 0.3.

In an additional embodiment, a crystalline polymorph of Compound I of claim I is crystalline polymorph Form C. In another embodiment, the crystalline polymorph exhibits an x- ray powder diffraction pattern having at least three, at least five, at least seven, or all, of the following peak positions at degree two-theta of about: 9.8±0.3, 10.2±0.3, 14.3±0,3, 17.4±0.3, 1 .2 . 0.3. 1 .9 - 0.3. 19.2 . 0.3. 22.1 ±0.3, 22.7±0.3, and 29.1±0.3. In still another embodiment, the crystalline polymorph exhibits an x-ray powder diffraction pattern having at least three, at least five, at least seven, at least ten, or all, of the following peak positions at degree two-theta of about: 9.0. 0.3. 9.8 - 0.3. 10.2 : 0.3. 14.3 . 0.3. 15.9 ±0.3, 1 7.4 - 0.3. 1 8.2 . 0.3. 18.9±0.3, 19.2±0.3, 19.6 . 0.3. 20.2 · 0.3. 21.3±0.3, 22.H0.3, 22.7±0.3, 24.7 . 0.3. 28.3 - 0.3. 28.9 : 0.3. 29.1 : 0.3. and 30.1±0.3.

In some embodiments, the crystalline polymorph of Compound I is polymorph Form D. In some other embodiments, the crystalline polymorph exhibits an x-ray powder diffraction pattern having peak positions at degree two-theta of about: 5.6±0.3, 26.0±0.3, and 26.7±0.3. In some additional embodiments, the crystalline polymorph exhibits an x-ray powder diffraction pattern having peak positions at degree two-theta of about: 8.5±0.3 and 19.3±0.3. In some further embodiments, the crystalline polymorph exhibits an x-ray powder diffraction pattern having at, least three, at least five, at least seven, at least ten, or all, of the following peak positio s at degree two-theta of about: 5.58, 7.43, 8.45, 9.21, 1 1.23, 11.98, 14.86, 17.025, 18.91, 22.80, 26.03, and 26.72.

In particular embodiments, the crystalline polymorph of Compound I is polymorph Form E, In other embodiments, the crystalline polymorph exhibits an x-ray powder diffraction pattern the peak positions at degree two-theta of about: 5.6±0.3, 14,4±0,3, and 23.5+0.3. In further embodiments, the crystalline polymorph exhibits an x-ray powder diffraction pattern the peak positions at degree two-theta of about: 18.9±0.3 and 21 ,0±0,3. In yet further embodiments, the crystalline polymorph exhibits an x-ray powder diffraction pattern having three or more, five or more, seven or more, ten or more, or all of, the peak positions at degree two-theta selected from the group consisting of about: 5.570, 6.846, 8.552, 9.505, 11.786, 14.374, 17.060, 17.431 ,

18.006, 18.893, 19.971 , 21.015, 22.347, 22.840, 23.528, 25.496, 26.393, 28.264, 29.563, 30.710, 32.263, and 34.045.

In still further embodiments, Compound I is in an amorphous form.

Additional embodiments include a combination of any of the foregoing embodiments and one or more pharmaceutically acceptable excipie ts. Other embodiments include a dosage form, such as a solid or semi-solid dosage form, comprising any of the foregoing crystal forms, amorphous forms, or combinations. In yet other embodiments, a dosage form comprising any of the foregoing crystal forms, amorphous forms, or combinations comprises one or more of a tablet, hard capsule, soft capsule, powder, suppository, and gel, or one or more of an injectable form, a transdermal patch, a sprayable form, and an implantable depot.

Other embodiments are a use of any of the foregoing embodiments in making a dosage form for inhibiting, or for inhibiting, a NGF receptor. Still other embodiments are a use of any of the foregoing embodiments in making a dosage form for treating a disorder, disease or condition selected from the group consisting of acute and chronic pain, cancer (e.g., pamcreatic cancer, gastric cancer, esophageal cancer, gastrointestinal cancer, colorectal cancer, lung cancer, liver cancer, brain cancer or human neuroblastoma, glioblastoma and meduUobiastoma,

retinoblastoma, leukemia, lymphoma, melanoma, malignant mesothelioma, breast cancer, bladder cancer, ovarian cancer, prostate cancer, thyroid cancer, squamous cell carcinomas), itching, atopic dermatitis, scabies, pityriasis, inflammatory bowel disease, inflammatory arthritis, asthma, human airway diseases, Chagas' disease, parasitic diseases, Alzheimer's, restenosis, atherosclerosis, psoriasis, thrombosis, skin diseases, inflammation, inflammation related diseases, or a disease, disorder or injury relating to dysmyelination or demyelination or the disease or disorder associated with abnormal activities of one or more Trk protein kinases, with therapeutic effective amount of the compound as described above, or a salt, solvate, or physiologically functional derivative thereof.

Surprisingly, the Compound Fs oral bio avail bility is dramatically increased to about 90 to 100% in rat, which represents a significant improvement, compared to about less than 20% oral bioavailability i rat of the tree acid Compound 701.

In another aspect, the invention provides pharmaceutical compositions comprising the compound described above, and a pharmaceutically acceptable vehicle.

In another aspect, the invention provides a method of use of Compound I in medical treament and prevention.

In another aspect, the invention provides a method of use of Compound I in medical treament and prevention of certain cancer (e.g., pamcreatic cancer, gastric cancer, esophageal cancer, gastrointestinal cancer, colorectal cancer, lung cancer, liver cancer, brain cancer or human neuroblastoma, glioblastoma and medulloblastoma, retinoblastoma, leukemia, lymphoma, melanoma, malignant mesothelioma, breast cancer, bladder cancer, ovarian cancer, prostate cancer, thyroid cancer and squamous cell carcinomas), itching, atopic dermatitis, scabies, pityriasis, inflammatory bowel disease, inflammatory arthritis, asthma, human airway diseases, restenosis, atherosclerosis, psoriasis, thrombosis, Chagas' disease, parasitic diseases,

Alzheimer's, pain, a disease, disorder, injury, or malfunction relating to dysmyelination or demyelination or the disease or disorder associated with abnormal activities of one or more Trk protein kinases, with combination of (a) therapeutic effective amount of the compound as described above, or a salt, solvate, or ester, prodrug, or physiologically functional derivative thereof, and either (bl ) an opioid analgesic or at least one analgesic agent that acts by a mechanism different from a. Trk antagonist or, (hi) an existing or approved anti-cancer agent or ehemotherapeutic or at least one existing or approved anti-cancer agent.

In another aspect, the invention provides a method for preparing Compound I described above.

Formu!a i

In another aspect, the invention provides most stable polymorphic forms of Compound I d preparation processes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 is a graph showing time-dependent pain relieving effect of Compound I (s.c.) in CFA-induced rat inflammatory pain.

FIGURE 2 is a graph showing oral dose-dependent reduction of neuropathic pain of the test article in rat model of neuropathic (CO) pain.

FIGURE 3 is IH-NMR spectra of the polymorph Form A of Compound I.

FIGURE 4 is X-ray powder diffraction (XRPD) spectra of the polymorph Form A of Compound I. FIGURE 5 is Raman spectra of the polymorph Form A of Compound 1.

FIGURE 6 is Differential Scanning Caiorimetry (DSC) spectra of polymorph Form A of Compound I.

FIGURE 7 is XRPD overlay polymorph Form A of Compound I at 40°C / 75% RH T = 0 (top) vs T = 7 days. FiGURE 8 is XRPD overlay polymorph Form A of Compound I at 80°C T = 0 (top) vs T = 7 days, FiGURE 9 is STA spectra polymorph Form A of Compound I.

FiG URE 10 is Raman spectra, overlay of the polymorph Form A of Compound I at T = 0 (bottom) vs T = 7 days (middle, 40°C / 75% RH) and T = 7days (top, 80°C).

FiGURE 11 is X-ray powder diffraction (XRPD) spectra of the polymorphic form. Form B of Compound I.

FiG URE 12 is STA spectra of the polymorphic form. Form B of Compound L

FiGURE 13 is DSC spectra of the polymorphic form, Form B of Compound I.

FIG URE 14 is X-ray powder diffraction (XRPD) spectra of the polymorphic form, Form C of Compound I.

FIGURE 15 is X-ray powder diffraction (XRPD) spectra of the amorphous Compound I.

FiGURE 16 is X-ray powder diffraction (XRPD) spectra of the polymorphic form, Form D of Compound 1.

FiGURE 17 is X-ray powder diffraction (XRPD) spectra of the polymorphic form, Form E of Formula I compound.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that singular prepositions such as "a," "an," and "the," are often used for convenience, however, all instances of the singular are intended to encompass the plural unless otherwise indicated either explicitly or from context. Further, it should be understood that all references, including journal articles, books, patents, technical documents, and the like, mentioned in this disclosure are hereby incorporated by reference in their entirety and for ail purposes.

Furthermore, all numerical data points should be understood to be modified by the term "about" as will be elaborated upon in the disclosure. Definitions

The term "physiologically functional derivative(s)" as used herein refers to any physiologically tolerated derivative of a compound of the present invention, for example, an ester or prodrug, which, upon administration to a mammal, e.g., a human, are transformed directly or indirectly to Compound I, or an active metabolite thereof. Physiologically functional derivatives include prodrugs of the compounds of the present invention. Examples of prodrug are described in H. Okada et al., Chem. Pharm. Bull. 1994, 42, 57-61 . Such prodmgs can be metabolized in vivo to a compound of the invention. These prodrugs may themselves be active or not.

Compounds of the present invention also include isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature. Examples of isotopes that may be incorporated into the compounds include, but are not limited to, Ή, Ή, ^" C, ^~* C, ^" N, Ό, O, etc. Compounds may exist in unsolvated forms as well as solvated forms, including hydrated forms and as N-oxides. In general, the salt, hydrated, solvated, and N-oxide forms are within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline forms or an amorphous form. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

"Patient" or "subject" includes, but is not limited to, animals such as, for example, mammals. Preferably, the patient is a human.

"Preventing" or "prevention" refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).

"Solvate" means a compound formed by solvation (the combination of solvent molecules with molecules or ions of the solute, i.e., a compound of the present invention), or an aggregate that consists of a. solute ion or molecule (the compound of the present invention) with one or more solvent molecules.

"Pharmaceutically acceptable" means suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use within the scope of sound medical judgment,

"Prodrug or softdrug" refers to a precursor of a pharmaceutically active compound wherein the precursor itself may or may not be pharmaceutically active but, upon administration, will be converted, either metabolically or otherwise, into the pharmaceutically active compound or drug of interest. For example, prodrug or softdrug is an ester or an ether form of a

pharmaceutically active compound. Several prodrugs have been prepared and disclosed for a variety of pharmaceuticals. See, for example, Bundgaard, H. and Moss, J., J. Pharm. Sci. 78: 122- 126 (1989). Thus, one of ordinary skill in the art knows how to prepare these precursors, prodrugs or softdrugs with commonly employed techniques of organic synthesis.

"Treating", "treat" or "treatment" of any disease or disorder refers, in some

embodiments, to ameliorating or preventing the disease or disorder (i.e., arresting, preventing, holding or reducing the development of the disease or at least one of the clinical symptoms thereof). In other embodiments "treating", "treat" or "treatment" refers to ameliorating at least one physical parameter, which may not be discernible by the patient. In yet other embodiments, "treating", "treat" or "treatment" refers to inhibiting, or holding or preventing the progress of, the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter) or both. In yet other embodiments, "treating", "treat" or "treatment" refers to delaying the onset of the disease or disorder.

"Therapeutically effective amount" means the amount of a compound that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the patient to be treated,

''Vehicle" refers to a diluent, adjuvant, excipient or carrier with which a compound is administered.

Reference will now be made in detail to preferred embodiments of the invention. While the invention will be described in conjunction with the preferred embodiments, it will be understood that it is not intended to limit the invention to those preferred embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit, and scope of the invention as defined by the appended claims. The phrases "an effective amount" and "an amount sufficient to" refer to amounts of a. biologically active agent that produce an intended biological activity.

The term "co-administer" or "co-administering" when used in reference to the administration of Trk (i.e., TrkA) antagonists and other agents indicates that the antagonist and other agent(s) are administered in a coordinated fashion so that there is at least some

chronological overlap in their physiological activity on the subject. Thus, a. TrkA antagonist can be administered simultaneously and/or sequentially with another agent. In sequential administration, there may even be some substantial delay (e.g., minutes or even hours or days) before administration of the second agent as long as the first administered agent is exerting some physiological effect on the organism when the second administered agent is administered or becomes active in the subject.

The term "reducing pain," as used herein, refers to decreasing the level of pain a subject perceives relati ve to the le vel of pain the subject would have perceived were it not for the intervention. Where the subject is a person, the level of pain the person perceives can be assessed by asking him or her to describe the pain or compare it to other painful experiences.

Alternatively, pain levels can be determined by measuring the subject's physical responses to the pain, such as the release of stress-related factors or the activity of pain-transducing nerves in the peripheral nervous system or the CNS, One can also determine pain levels by measuring the amount of a well-characterized analgesic required for a person to report that no pain is present or for a subject to stop exhibiting symptoms of pain. A reduction in pain can also be measured as an increase in the threshold at which a subject experiences a given stimulus as painful. In certain embodiments, a reduction in pain is achieved by decreasing "hyperalgesia," the heightened sensitivity to a noxious stimulus, and such inhibition can occur without impairing "nociception," the subject's normal sensitivity to a "noxious" stimulus.

As used with reference to pain reduction, "a subject in need thereof refers to an animal or person, preferably a person, expected to experience pain in the near future. Such animal or person may have an ongoing condition that is causing pain currently and is likely to continue to cause pain. Alternatively, the animal or person has been, is, or will be enduring a. procedure or event that usually has painful consequences. Chronic painful conditions such as diabetic neuropathic hyperalgesia and collagen vascular diseases are examples of the first type; dental work, particularly that accompanied by inflammation or nerve damage, and toxin exposure (including exposure to ehemotherapeutic agents) are examples of the latter type.

"Inflammatory pain" refers to pain arising from inflammation. Inflammatory pain often manifests as increased sensitivity to mechanical stimuli (mechanical hyperalgesia or tenderness). For examples, inflammatory pain is due to a condition selected from the group consisting of: burn, sunburn, arthritis, colitis, carditis, dermatitis, myositis, neuritis, mucositis, urethritis, cystitis, gastritis, pneumonitis, and collagen vascular disease.

"Neuropathic pain" refers to pain arising from conditions or events that result in nerve damage. "Neuropathy" refers to a disease process resulting in damage to nerves. "Causalgia" denotes a state of chronic pain following nerve injury. "Allodynia" refers to a condition in which a person experiences pain in response to a normally nonpainful stimulus, such as a gentle touch. For examples, neuropathic pain is due to a condition selected from the group consisting of: causalgia, diabetes, collagen vascular disease, trigeminal neuralgia, spinal cord injury, brain stem injury, thalamic pain syndrome, complex regional pain syndrome type I/reflex sympathetic dystrophy, Fabry's syndrome, small fiber neuropathy, cancer, cancer chemotherapy, chronic alcoholism, stroke, abscess, demye!inating disease, viral infection, anti-viral therapy, AIDS, and AIDS therapy. Neuropathic pain is due to an agent selected from the group consisting of:

trauma, surgery, amputation, toxin, and chemotherapy.

As used herein, the term "generalized pain disorder" refers to a group of idiopathic pain syndromes (e.g., fibromyalgia, irritable bowel syndrome, and temporomandibular disorders), for which the pathogenic mechanism is currently unknown, characterized by diffuse or generalized pain, and for which a diagnosis of inflammation or neuropathy as the direct cause of pain is excluded.

An "analgesic agent" refers to a molecule or combination of molecules that causes a reduction in pain.

The difference between "acute" and "chronic" pain is one of timing: acute pain is experienced soon (e.g., generally within about 48 hours, more typically within about 24 hours, and most typically within about 12 hours) after the occurrence of the event (such as

inflammation or nerve injury) that led to such pain. By contrast, there is a significant time lag between the experience of chronic pain and the occurrence of the event that, led to such pain. Such time lag is generally at least about 48 hours after such event, more typically at least about 96 hours after such event, and most typically at least about one week after such event.

The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as w ^r ell as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such term in relation to pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the

pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, for example, leukemia, lymphoma, blastoma, carcinoma and sarcoma. More particular examples of such cancers include chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL), squamous cell carcinoma, small- ceil lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, malignant hepatoma, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, breast cancer, colon carcinoma, and head and neck cancer, gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer, multiple myeloma, acute myelogenous leukemia (AML), and chronic lymphocytic leukemia. (CML).

It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a compound" includes a combination of two or more compounds or molecules, and the like.

In one aspect of the present invention, a compound has the following structure:

Sodium 4~((3-{4-cyciohexylpiperazin- 1 -yl)-6-oxo-6H-anthra[ 1 ,9-cd]isoxazol

y l)amino)benzoate .

The present invention includes amorphous form of Compound I as well as various polymorphic forms of Compound I. Polymorphism can be characterized as the ability of a compound to crystallize into different crystal forms, while maintaining the same chemical formula. A crystalline polymorph of a given drug substance is chemically identical to any other crystalline polymorph of that drug substance in containing the same atoms bonded to one another in the same way, but differs in its crystal forms, which can affect one or more physical properties, such as stability, solubility, melting point, bulk density, flow properties,

bioavailability, etc.

In one embodiment, the crystalline forms are characterized by the interlattice plane intervals determined by a X-ray powder diffraction pattern. The spectrum of XRD is typically represented by a diagram plotting the intensity of the peaks versus the location of the peaks, i.e., diffraction angle 2Θ (two-theta) in degrees. The intensities are often given in parenthesis with the following abbreviations: very strong = vst: strong = st; medium = m: weak = w: and very weak = vw. In one embodiment, the intensity of about 81% to 100% is very strong; the intensity of about 61% to 80% is strong; the intensity of about 41% to 60% is medium; the intensity of about 21% to 40% is weak; and the intensity of about 1% to 20% is very weak. The

characteristic peaks of a given XRD can be selected according to the peak locations and their relative intensity to conveniently distinguish this crystalline structure from others. Those skilled in the art recognize that the measurements of the XRD peak locations and/or intensity for a. given crystalline form of the same compound will vary within a margin of error. The values of degree 2Θ allow appropriate error margins. Typically, the error margins are represented by "±". For example, the degree 2Θ of about "9.7±03" denotes a range from about 9.7+0.3, i .e., about 10.0, to about 9.7-0.3, i.e., about 9.4. Depending on the sample preparation techniques, the calibration techniques applied to the instruments, human operational variation, and etc, those skilled in the art recognize that the appropriate error of margins for a XRD can be ±0.5; ±0.4; ±0.3; ±0.2; ±0.1 ; ±0.05; or less.

The term "substantially similar" as used herein means an analytical spectrum, such as XRD pattern, Ή- MR spectrum, FT-IR spectrum, Raman spectrum, TGA thermogram, etc., which resembles the reference spectrum to a great degree in both the peak locations and their intensity.

In one specific embodiment, a process for the preparation of Compound I, comprising the following steps:

Formii!a ! Preparation of Intermediate 5. Mixing 4-amino benzoic acid and lithium hydroxide in DMAc, then adding 3,5-dibromo-6H-anthra[l ,9-cd]isoxazol-6-one into the mixture and raising the temperature to about 45°C to 55°C. Stirringing the reaction mixture for 18-20 hours. Slowly adding MTBE to the reaction mixture and then cooling to 0 to 10°C. Filtering the solid and drying at room temperature to get 4-((3-bromo-6-oxo-6H-anthra[ 1 ,9-cd]isoxazol-5- yl)amino)benzoic acid (Intermediate 5).

Preparation of Intermediate 6, Dissolving 4-((3-bromo-6-oxo-6H-anthra[ 1 ,9-cd]isoxazol-5- yl)amino)benzoic acid in DMSO, then adding triethylamine and 1-cyclohexyl piperazine (2a) into the solution. Raising temperature to 60-70°C. After 2-3 hours, slowly adding MTBE and MeOH solution and cooling to room temperature. Isolation and washing of the wet cake with MTBE and MeOH followed by filtering provided 4-((3-(4-cyelohexylpiperazin- 1 -yl)-6-oxo-6H- anthra[.l ,9-cd]isoxazol-5-yl)amino)benzoic acid (Intermediate 6) as a solid.

Preparation of Compound L Dissol ving intermediate 6, 4-((3-(4-cyclohexylpiperazin-l -yl)-6- oxo-6H-anfhra[I ,9-cd]isoxazol-5-yl)ammo)benzoic acid in sodium hydroxide in methanol solution. Raising temperature to about 40°C and maintaining for 2-3 hours. Then lowering temperature and filtering solid to get Compound I, sodium 4-((3-(4-cyclohexylpiperazm-l-yl)-6- oxo-6H-anthra[l ,9-cd]isoxazol-5-yl)amino)benzoate. The compound was purified in sodium hydroxide in methanol solution and dried. It gave more than 99% (HPLC, area%) purity and about 45% yield. Mass spectra gave [M+l] = 523.2. Ή-NMR (400 MHz, DMSO-d6, see also Figure 3), ppm (δ): 11.79 (1H, s), 8.48 (1H, d), 8.20 (1H, d), 7.93 (2H, d), 7.84 (1H, t), 7.72 (1H, t), 7.35 (2H, d), 6.39 (1H, s), 3.85 (4H, m), 2.72-2.70 (4H, m), 2.28-2.265 (1H, m), 1.72-1.78 (4H, m), 1.55-1 .58 (1H, m), 1.08-1.23 (5H, m).

In another aspect of the present invnetion, crystalline polymorphs of Compound I are studied and synthesized.

In one of crystalline polymorphs, Form A of Compound I, wherein it is characterized by

X-ray Powder Diffraction (XRPD) having one or more characteristic peak positions of 9.7±0.3, 10.0 . 0.3. 14.5 - 0.3. ! . 0.3. 18.1±0.3, 20.1 0.3. 2 ! .2 . 0.3. and 23.6 . 0.3 degree 2-theta.

The crystalline polymorph Form A of Compound I is further characterized by XRPD having one or more peak positions of 7.0±0.3, 8.6±0.3, 9.7±0.3, 10.fe0.3, 12.3+0.3, 14.5±0.3, 15.1±0.3, I 7.5 . 0.3. 18.1±0.3, 19.0±0.3, 20.1±0.3, 21.2±0.3, 22.4. 0.3. 23.6 : 0.3. 24.6 : 0.3.

25.6+0.3, 26.5±0.3, 28.2+0.3, 29.4±0.3, 30.6±0.3, 34.1+.3 and 35.0±0.3 degree 2-theta. In another aspect, the present invention comprises another crystalline form (Form B) of the Compound I described above, XRPD peaks at one or more of 9,8±0,3, 10.2±03, 14.5±0.3, 17.8+0.3, i 8 : 0.3.. 19.6±0.3, 21.0±0.3, 21.7±0.3, 23.1+0.3, degree 2-theta.

In another aspect, the present invention comprises another crystalline form (Form B) of the Compound I described above, XRPD peaks at one or more of 9.8±0.3, I0.2±0.3, 14.5+0.3, 17.8±0.3, 1 .5 - 0 3. 19.6±0.3, 21.0±0.3, 21 .7±0.3, 23.1±0.3, 25.0 - 0.3. 25.6±0.3, 28.4±0.3, 29.4+0.3, 30.2+0.3, 31.6+0.3 degree 2-theta. An example of Form B is given in Figure 1 1. Form. B can be a hydrate or hemi-hydrate polymorphic form, however, experimental results are consistent with the proposition that Form B does not, conver to Form A. upon simple heating or drying.

In another aspect, the present invention comprises a third crystalline form (Form C) of the Compound I described above, XRPD peaks at one or more of 9.8+0.3, 10.2+0.3, 14.3+0.3, 17.4+0.3, 18.2+0.3, 18.9+0.3, 19.2+0.3, 22.1+0.3, 22.7+0.3, 29.1+0.3, degree 2-theta.

In another aspect, the present invention comprises a third crystalline form (Form C) of the Compound I described above, XRPD peaks at one or more of 9.0+0.3, 9.8+0.3, 10.2+0.3, 54.3+0.3, 15.9 ±0.3, 17.4+0.3, 18.2+0.3, 18.9+0.3, 19.2+0.3, 19.6+0.3, 20.2+0.3, 21 .3+0.3, 22.1 +0.3, 22.7+0.3, 24.7+0.3, 28.3+0.3, 28.9+0.3, 29.1+0.3, 30.1+0.3 degree 2-theta. An example of Form C is given in Figure 14.

In another aspect, the present invention comprises a third crystalline form (Form D) of the Compound I described above, XRPD peaks at one or more of 5.6+0.3, 8.5+0.3, 14.9+0.3, 17.0+0.3, 26.0+0.3, 26.7+0.3, degree 2-theta.

In another aspect, the present invention comprises a third crystalline form (Form D) of the Compound I described above, XRPD peaks at one or more of 5.6+0.3, 7.4+0.3, 8.5+0.3, 9.2+0.3, 1 1.2±0.3, 12.0+0.3, 14.9 +0.3, 17.0+0.3, 18.9+0.3, 22.8+0.3, 26.0+0.3, 26.7+0.3, degree 2-theta. An example of Form D is given in Figure 16.

An amorphous form of Compound I was also found and characterized by XRPD. An example of amprpjous of Compound I is given in Figure 15.

In another aspect, the present invention comprises a method of producing the crystalline form of Compound I described above.

Example 1 Preparation of 4-((3-bromo-6-oxo-6H-antfara[ 1 ,9-cd]isoxazoi-5-yl)amino)benzoic acid. About 70g of 4-amino benzoic acid and 22g of lithium hydroxide were mixed in 800niL of DMAc. Then lOOg of 3,5-dibromo-6H-anthra[l,9-cd]isoxazol-6-one was dissolved in the pre-mixed solution. Nitrogen protection was applied to the reaction mixture at 45-55°C for about 18-20 hours. After reaction completion was confirmed by HPLC, 30mL of MTBE was slowly added to the reactor. Reaction mixture was then slowly cooled to 0-10°C under nitrogen protection.

Centrifuged the solid and washed with 18mL of MTBE. Dried the wet cake under 25-30°C to obtain about 69g of 4-((3-bromo-6-oxo-6H-anthra l ,9-cd]isoxazol-5-yi)amino)benzoic acid. Example 2

Preparation of 4-((3-(4-cyclohexylpiperazin- 1 -yl)-6-oxo-6H-anthra[ 1 ,9-cd]isoxazol-5- yl)amino)benzoic acid. About 70g of 4-((3-bromo-6-oxo-6H-anthra[l,9-cd]isoxazol-5- yl)amino)benzoic acid was dissolved in 950mL of DMSO. About 50mL of TEA and 50g of 1- cyciohexyl piperazine were added to the reaction. Temperature was raised to 60-70°C. After 2-3 hours, slowly added 500mL of MTBE and MeOH (10: 1) solution and adjusted the temperature to 40-50°C. The solid was centrifuged and washed by lOOmL of MTBE and MeOH solution and followed by 1 OOmL of MeOH. Solid was dried under reduced pressure at 25-30°C for 12-24 hours to obtain the 4-((3-(4-cyclohexylpiperazin-l-yl)-6-oxo-6H-anthra[l,9-cd]is oxazol-5- yl)amino)benzoic acid at about 98% purity and about 91% yield.

Example 3

yl)ammo)benzoate. About 80g of 4-((3-(4-cyclohexylpiperazin- 1 -yl)-6-oxo-6H-anthra[ 1 ,9- cd]isoxazol-5-yl)amino)benzoic acid was slurred in 2500mL of 0.4 M NaOH/MeOH between 40-45°C for 2-4 hours. After confirmed the reaction completion by HPLC, slowly cooled the reaction to room temperature. The solid was centrifuged and washed with 20mL of MTBE. The wet cake was re-suspended in about 1 OOOmL of 0.1M aOH in MeOH solution under room temperature. It was centrifuged and washed with 224mL of MTBE again. The filtered solid was suspended in 996mL of MTBE under room temperature for 1-2 hours. The solid was separated and dried at 25-30°C under pressure for 52-24 hours to obtain the final desired product with purity more than 99% and about 90% yield. Mass spectra give [M+1J - 523.2. ^! H-NMR (400 MHz, DMSO-d6, see Figure 3), ppm (δ): 11 ,79 (IH, s), 8,48 (IH, d), 8.20 (IH, d), 7.93 (2H, d). 7.84 (I H, t), 7.72 (IH, t), 7.35 (2H, d), 6.39 (IH, s), 3.85 (4H, m), 2.72-2.70 (4H, m), 2.28-2.265 (IH, m), 1.72-1.78 (4H, m), 1.55-1.58 (IH, m), 1.08-1.23 (5H, m). Sodium content is 3.8%. The Raman spectrum is shown in Figure 5. The XRPD, see Table below and Figure 4, confirmed the polymorphic form.

Table 1. XRPD Table of the polymorphic form, Form A of Compound L

Angle d-value Intensity

2-Theta° (Angstrom) (%) ^"

1 7.160 12.3354 14.3

2 8.757 10.0899 22.4

3 9.820 9.0001 70.1

4 10.161 8.6986 88.1

5 10.522 8.4006 7.3

6 12.459 7.0985 14.5

"7 14.641 6.0454 82.0

8 15.219 5.8170

9 17.680 5.0124 41.0

10 18.240 4.8598 73.0

1 1 19.104 4.6420 14.2

12 20.220 4.3883 100.0

53 21.381 4.1525 33.3

14 ^■ -) ^■ -) <; "7(j 3.9347 50.1

15 23.721 3.7478 99.7

16 24.898 3.6733 1 1.8

17 25.761 3.4555 17.8

18 25.522 3.3581 13.6

19 27.565 3.2804 57.7

20 28.321 3.1487 10.5

21 29.481 3.0273 12.8

-> -> 29.781 2.9976 8.7

23 30.478 2,9306 7.7

24 30.925 2.8896 58.6

25 34,281 2.6137 12.1

26 35.120 2.5532 8.0

· ^■ > - 35.483 2.5279 6.0

Accelerated stability tests of Compound Ϊ at 40°C / 75% RH and 80°C detected no instability as measured by XRPD and Raman over 1 week, see Figure 7 and Figure 8 and Figure 50. Thus, Form A is a stable polymorphic form which is particularly useful for pharmaceutical compositions.

STA data (Figure 9) indicated the Compound I was not hydrated or solvated. The DSC data did not indicate a clear melt up to about 200° C, as shown in Figure 6. Form B was prepared by using slurry method from either amorphous Compound I or other polymorphic forms with solvent of ethanol: water (9: 1), which was followed by drying under reduced pressure and ambient temperature.

Form C was prepared by using slurry method from either amorphous Compound I or other polymorphic forms with solvent of THF: water (7:3), which was followed by drying under reduced pressure and ambient temperature.

Form. D was prepared by using methanol recrystallization process from either amorphous Compound I or other polymorphic forms, which was followed by drying under reduced pressure and ambient temperature.

Form E was prepared from either amorphous Compound I or other polymorphic forms by using slurry with the compound of Formula I in 0.1 M NaOH / methanol solution, followed by- washing with MTBE, which was followed by air dry.

Form D of the compound of Compound I can have an XRPD spectrum as shown in Figure 16, for example, with one or more, three or more, five or more, seven or more, ten or more, or all of the peaks sho wn in Table 2.

Table 2

XRPD Table of the polymorphic form, Form D of Compound 1

In another aspect, the present invention comprises another crystalline form (Form E) of the Formula I compound described above. Form E can exhibit an XRPD pattern with peaks at one or more of 5.6±0.3, 6.8+0.3, 8.6+0.3, 9.5±0.3, 1 1.8±0.3, 14.4+0.3, 17.1+0.3, 17.4 : 0.3. 18.0 ±0.3, 18.9±0.3, 20.0+0.3, 21.0±0.3, 22.3±0.3, 23.5±0.3, 25.5+0.3, 26.4+0.3, 28.3±0.3, 29.6+0.3, 30.7+0.3, 32.3+0.3, and 34.0+0.3 degrees 2-t eta, such as the XRPD peaks described in Table 3. Figure 17 shows an XRPD diffraction pattern of Form E.

Table 3

To prepare a pharmaceutical composition containing the active substance, particularly an orally administered pharmaceutical composition, most preferably a tablet, procedures known in the art may be used. For example, tablets can be prepared according to the formulations and methods discussed in Remington: The Science and Practice of Pharmacy, which is hereby incorporated by reference for all purposes. Suitable tablets may be obtained, for example, by mixing the active substance(s) with known excipients, for example, inert diluents such as mannitol, sorbitol, xylitol, saccharose, calcium carbonate, calcium phosphate, or lactose, disintegrants such as croscarmellose sodium salt ( cellulose carboxymethylether sodium salt, cross-linked), crospovidone, sodium, starch glycolate, hydroxypropylcellulose (low-substituted), or maize starch, binders such as polyvinylpyrrolidone, copolymers of vinylpyrrolidone with other vinyl derivatives (copovidone), hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, or starch, lubricants such as magnesium stearate, sodium stearyl fumarate, or talc and/or agents for obtaining delayed release, such as

hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose acetate phthalate, or polyvinyl acetate. The tablets may also comprise several layers. The following are some examples of pharmaceutical preparations which may be used according to the invention. They are intended purely as illustrations by way of example without restricting the subject matter of the in vention thereto. AH of the present crystalline polymorphs, i.e., Forms A, B, C, D, and E, can be used for pharmaceutical compositions as exemplified in the formulation examples below.

FORMULATION EXAMPLE 1

Tablet 1

Ingredients mg

Total 500,000

FORMULATION EXAMPLE 2

Tablet 2

FORMULATION EXAMPLE

Tablet 3

FORMULATION EXAMPLE 4

By directly compressing the Compound 1 with the excipients sorbitol and magnesium stearate, tablets are obtained whose concentration of active substance corresponds to an amount of 80 mg, 40 mg, and 20 mg of free acid of Compound I. Tablet Containing the Equivalent of 80 mg of Free Acid of Compound I.

Tablet Containing the Equivalent of 20 mg of Free Acid of Compound I.

FORMULATION EXAMPLE 5

The Compound I is first mixed with mannitol, red iron oxide and hydroxypropylcellulose in an intensive mixer. Then magnesium stearate is added by sifting through a 0.8 mm screen and the mixture is subjected to dry granulation in a roller compactor. In parallel, hydrochlorothiazide is mixed with mannitol, macrocrystalline cellulose, sodium glycol starch, and red iron oxide in an intensive mixer. Both this mixture and the granulated Compound I are sieved through a 0.8 mm screen, mixed together in a free fail blender, and finally subjected to a last mixing process with magnesium stearate screened through a 0.8 mm screen. A composition is obtained which can be compressed without any problems and the tablets produced from it exhibit good solubility for the active substances. This composition of active substances and excipients is compressed with a suitable tablet press. Tablets of the following composition are prepared, the amount of

Compound I contained in each tablet corresponding to an amount of 80 nig of the free acid of Compound I.

Total 100.000 1 6.667 100.000

Hydrochlorothiazide 12.500 2.083

Mannitol 325.500 54.250

Cellulose microcrystalline 120.000 20.000

Sodium glycol starch 30.000 5.000

Red iron oxide 0.500 0.083

Magnesium stearate 1 1.500 1.917

Total 600.000 100.000

FORMULATION EXAMPLE 6

Hydrochlorothiazide, Compound I, sorbitol, and red iron oxide are mixed in a free fall blender, passed through a 0.8 mm screen and, after the addition of magnesium stearate, processed in a free fall blender to form a powdered mixture. This composition of active substances and excipients

is then compressed into tablets using a suitable tablet, press. Tablets of the following composition are prepared, the amount of Compound I contained in each tablet corresponding to an amount of 80 mg of the free acid of Compound I.

FORMULATION EXAMPLE Capsule Containing the Equivalent of 1 00 mg of Free Acid of Compound I.

X-ray Powder Diffraction (XRPD . Approximately 2 mg of sample was gently compressed on the XRPD zero back ground single obliquely cut silica sample holder. The sample was then loaded into a D/MAX 2200 X-ray powder diffractometer (Rigaku) or a Philips X-Pert MPD diffractometer and analyzed using the following experimental conditions (Tube anode: Cu; Generator tension: 40 kV; Tube current: 40 mA; Wavelength alphal : 1.54056 A; Wavelength alpha2: 1.5444 A; Start angle [2 tiieta]: 5; End angle [2 theta]: 50; and Continuous scan). For suspected novel forms a slightly slower scan speed was used over a range of 4 - 40°2θ.

It is known in the art that for the same sample or test article measured by different X- ray powder diffractometers or, even by the same X-ray powder diffractometer but measured at different times or temperature or conditions, the values of peak positions at degree two-theta may have about ±0.3 or ±0.5 variations.

Raman spectroscopy. Samples were analyzed by a Nicolet Almega XR Dispersive Raman Microscope for its Raman spectrum using the following conditions (Exposure Time: 1 .0s;

Acquisition No: 10; Pinhole Size: 25, 50 or 100 μηι; Wavelength range: 2000~300cm ^"] (single grating); Laser: He-Ne 780nm 100% power; Objective: 20x/0.40 or 50x/0.75

(magnifier/numerical aperture number)). Then the measured Raman spectra were corrected b - baseline subtraction using the software OMNIC™ v7.3.

Simul taneous Thermal Anal y si s (ST A) . Approximately 5 mg of sample was accurately weighed into a ceramic crucible and it was placed into the chamber of Perkin-Elmer STA 600 TG A/DTA analyzer at ambient temperature. The sample was then heated at a rate of 10°C/min from 25°C to 300°C during which time the change in weight was monitored as well as DTA signal. The purge gas used was nitrogen at a flow rate of 20cm ³ /min.

Differential Scanning Calorimetry (DSC). Approximately; 5 mg of each sample was weighed into an aluminum DSC pan and sealed non-hermetically with an aluminum lid. The sample was then loaded into a Perkin-Elmer jade DSC and held at 25°C. Once a stable heat- flow response was obtained, the sample was then heated to 300°C at a scan rate of 10°C/min and the resulting heat flow response was monitored, A 20 em'/min helium purge was used. Prior to analysis, the instrument was temperature and heat flow verified using an indium standard.

Polarised Light Microscopy (PLM). An Olympus BX50 microscope, equipped with an analyser and polariser, was used to observe each sample under polarised light. Micrographs of the sample were taken by using a JVC-TKC1380 digital camera connected to a PC running Studio QuickStart version 9.3.2. A. 20*/0.5 (magnifier/numerical aperture (NA) value) objective was used to view samples and capture images.

Gravimetric Vapor Sorption (GV8) . Approximately 20 mg of sample was placed into a wire-mesh vapor sorption balance pan and loaded into an 'IgaSorp' vapor sorption balance (Hiden Analytical Instruments). The sample was then dried by maintaining a 0% humidity environment until no further weight, change was recorded. Subsequently, the sample was then subjected to a ramping profile from 0 - 90 % RH at 10 % RH increments, maintaining the sample at each step until equilibration had been attained (99% step completion). Upon reaching equilibration, the % RH within the apparatus was ramped to the next step and the equilibration procedure repeated. After completion of the sorption cycle, the sample was then dried using the same procedure. The weight change during the sorption/desorption cycles were then monitored, allowing for the hygroscopic nature of the sample to be determined.

BIOLOGICAL ACTIVITIES

Rat Pharmacokinetics. Compound I was dosed to nonfasted male CD IGS rats (weighting 180- 250 g, Charles River Laboratories) by single intravenous (IV, n=3/group) or oral gavage (PO, n=4/group, including control rat group for drag-free blood and brain collection) administration at a nominal dose levels of 3 mg/kg (IV) or 5 mg/kg (IV) and 10, 50, 100 mg kg (PO) respectively. Compound was formulated in either DMSO/Soiutol® HS 15/phosphate buffered saline, pH 7.4 (PBS) for both IV and PO dosing, or 10% (w/v) Povidone 12 in water for IV dosing or 10% (w/v) Povidone K25 in water for PO dosing, or 18% (w/v) CrosPovidone in water for PO dosing. An intravenous profile was obtained by taking serial or terminal blood samples at 3, 10, 30, 60, 120, 240, 360, 1440 min post dose. An oral profile was obtained by taking serial or terminal blood samples at 10, 30, 60, 120,240,360,480, 1440 min post dose. Plasma Sample Collection from Rats. Animals were sedated under general inhalant anesthesia (3% isofhirane) for blood collection by cardiac puncture. Blood aiiquots (300-400 iL) were collected in tubes coated with lithium heparin, mixed gently, then kept on ice and centrifuged at 2,500 xg for 15 minutes at 4°C, within 1 hour of collection. For control animals, blood was collected by cardiac puncture. The plasma was then harvested and kept frozen at - 20°C until further processing. Plasma Sample Collection from Cannulated Rats. Blood collection was carried out from the jugular vein catheter. Blood aiiquots (300 - 400 μΐ,.) were collected in tubes coated with lithium heparin, mixed gently, then kept on ice and centrifuged at 2,500 xg for 15 minutes at 4°C, within 1 hour of collection. For control animals, blood was collected by cardiac puncture. The plasma was then harvested and kept frozen at -20°C until further processing.

Brain Sample Collectio from Rats. Immediately after the blood sampling, rats were decapitated and the whole brains were quickly removed, rinsed with cold saline (0.9% NaCl, g/mL), surface vasculature ruptured, blotted with dry gauze, weighed and kept on ice until further processing within 1 hour of collection. Each brain was homogenized in 3 mL cold phosphate-buffered saline, pH 7.4 for 10 seconds on ice using Power Gen 125, The brain homogenate from each brain was then stored at -20°C until further processing.

Plasma and brain samples were subjected to quantitative analysis by LO S/MS using compound-specific mass transitions. Drug concentration-time profiles were generated and non- compartmental P analysis (using W inNonl in) used to generate estimates of half-life ( ), clearance, volume of distribution (V _ss ), and oral bioavailability (F%) (Gabriesson, J. and Weiner, D. Pharmacokinetic and Pharmacodynamic Data Analysis: Concepts and Applications. Swedish Pharmaceutical Press. 1997). The concentrations of the test compound in brain (ng/g brain tissue) and in plasma (ng/mL) as well as the ratio of the brain concentration and the plasma

concentration at each time point were determined and reported.

For Compound I, the mean pharmacokinetic parameters are determined as following:

Dose Route Dose Time Brain Plasma Ratio

(rng/kg) (mg/kg) (min) concentration Concentration Brain/Plasma

(ng/g brain) (ng/mL) (mL/g brain)

3 IV 5 30 153 11279 1.4%

3 IV 5 60 120 8161 1.8%

3 IV 5 180 23 1833 1.9%

Compound inhibition in a live, whole cell based functional assay: There are several methods to measure whole length TrkA activation stimulated by its natural ligand or agonist NGF in live ceils. For example, the PathHunter Profiling services offered by DiscoveRx (Fremont, CA). The PathHunter technology is an adaptation of enzyme fragment complementation that provides a novel, generic functional cell-based assay format for detecting protein-protein interactions. In this cell-based assay approach, with U20S ceil background, a small peptide epitope (P ) is expressed recombinantly on the intracellular C-terminus of TrkA (human full length). This is co- expressed with a larger sequence, termed enzyme acceptor (EA) that is attached to a cytoplasmic protein SHC1 which will interact with TrkA intracellular!}'. NGF induced activation of TrkA receptor causes either homo- or hetero-dimerization of TrkA resulting in cross-phosphorylation. The SHCI-EA fusion protein then binds the phosphorylated TrkA receptor forcing

complementation of the PK and EA fragment. This interaction generates an active beta- galactosidase enzyme, which is detected using a chemiiuminescent substrate. In such cell-based functional assays, Compound C of the present invention inhibits NGF stimulated TrkA activation at low nanomolar concentration (cellular IC ₅₀ is about 50 nM, mean of triplicate), while virtually has no effect on either BDNF stimulated TrkB, or NT3 stimulated TrkC activation (IC ₅₀ more than 10,000 nM in both cases, triplicate, with a positive control compound of pan-kinase inhibitor, staurosporine or K~252a, an internal agonist control and a negative control compound). Cell Viability and Proliferation Assays. To assess the chemosensitivity of tumor cells, cell viability is measured by CellTiter-Glo® Luminescent Cell Viability Assay (Promega; WI, USA) per the manufacturer's instruction. Briefly, 5 x 103 to 7 ^x 105 cells/ml are cultured in sterile 96- well plates in the presence of increasing concentrations of the drugs (test article, 0 to 500 μ,Μ), or vehicle in RPMI medium. The plates are then incubated for 24 to 96 h, and then 100 μΐ of CellTiter-Glo reagent is added to lyse the cells. After a 10-min incubation at room temperature, the luminescence is recorded in a luminometer with an integration time of 1 s per well. The luminescence signals for the drug-treated ceils are normalized by the luminescence signal obtained from vehicle -treated ceils. As an alternative method to quantitate cell viability, the trypan blue exclusion dye method was used. Vehicle- or drug-treated cells were assayed by adding trypan blue solution (0.4% in phosphate-buffered saline [PBS]) to the culture medium. After 3 mm, the number of dead cells that retained the dye is compared to the total number of cells to calculate cell viability. GrapbPad Prism 5 software is used to calculate 1C50 and plot, effect-dose curve of drugs. Multiplate reader; EnVision 2104 (PerkinElmer). % of control variability = 100*[(X(dru.g_treated) - L(baseline))/(H(vehicle__control) - L(baseline))]. Such assays show that for example. Compound I, has IC50 value about 2 to 5 μΜ in human pancreatic cancer cells (from ATCC) of AsPC-1 , MIA PaCa-2, BxPC-3, Capan-1 and Panc-1 ; about 2 to 8 μ,Μ in human liver cancer cells of SK-HEP-1 and HepG2; and about 7 μΜ in human stomach cancer cells of NCI-N87, after 24 h or 48 h or 96 h incubation. Taxol, erlotinib, sorafenib and gemcitabine are used as controls, and the compounds of present inventions are synergistic or additive with gemcitabine, taxol, erlotinib or sorafenib in pancreatic or liver cancer cells.

Rat CFA-induced Inflammatory Pa o. Model. Hyperalgesia was induced by subcutaneously injecting 50 μΐ, of CPA (Sigma- Aldrich, St. Louis, MO, USA) into the plantar surface of the left hind paw of the rats using a 30-gauge hypodermic needle under sevoflurane anesthesia. The classical signs of inflammation, including edema and redness, for up the last day of tests were recorded. To assess the effect of test article on CPA-induced inflammatory pain, the rats were anesthetized with sevoflurane 3 hours after CPA injection and then injected 50 μΐ. of either 1 mM test article (n = 6) or Saline (n = 4) subcutaneously (S.C.) into the same site as the CPA injection, using a 30-gauge hypodermic needle under sevoflurane anesthesia, according to published method (Ueda ., et al, J Pharmacol. Sci., 2010; 112(4):438-43). Paw withdrawal latencies (thermal hyperalgesia) are measured before and at 2 and 4 and 2, 4, and 7 days after CPA injection. Mechanical thresholds were also measured in the same way with either Formula 1 compound (n = 6) or Saline (n = 4) at similar different time points (for example, 1, 3 hour and 3 days). The noxious heat and mechanical thresholds were separately measured in each group of rats. The threshold was measured 3-5 times in each rat and then averaged. Stimulus interval was

5 min. The analgesic effect of Compound I on this rat CPA-induced inflammatory pain model is given in Figure 1 , where it clearly shown that Compound I is efficacious in relieving the pain in this rat pain model.

Chronic Constriction injury (CCI) Model of Neuropathic Paisi in Rat The CCI model is one of the most commonly used mono -neuropathic pain model firstly described in details by Bennett and Xie (Bennett GJ, Xie YK. Pain. 1988;33(1):87-107). It mimics important clinical chronic pain symptoms such as mechanical allodynia and thermal hyperalgesia. Chronic constriction injury of the sciatic nerve was produced by tying four loose ligatures around the left, sciatic nerve according to the method of Bennett and Xie. This procedure resulted in tactile allodynia in the left hindpaw. Calibrated von Frey filaments were used to determine the lowest, mechanical (tactile) threshold required to evoke a brisk paw withdrawal reflex in the rat hindpaws. Rats were allowed to acclimatize in wire mesh cages for 15-20 min prior to von Frey testing. Assessment of paw withdrawal thresholds (PWTs) using von Frey filaments was undertaken prior to CCI-surgery (pre-surgery baseline on day 0). Before the drug dosing on day 14, the pre-dose baseline was recorded for each rat. Rats were included in the study only if they did not exhibit motor dysfunction (e.g., paw dragging or dropping) and their PWT was below to 4 g. Drug-naive CCI-rats (n = 4 - 6 per group) were used. The oral (PC ⁾ ) gavage vehicle was either 10% (w/v) Povidone K25 in water for PC) dosing, or 58% (w/v) CrosPovidone in water for PC ⁾ dosing, or 0.5% CMC-Na/ ^' 0.1% Tween 80 in distilled water for PO dosing. The positive control gabapentin was dissolved in the vehicle and orally given at 100 mg/kg (by oral gavage). Test article was dissolved or suspended in the vehicle and orally given at 25, 50, 100 and 150 mg/kg. Each CCI-rat was administered a single oral dose of test article, gabapentin or vehicle control , 2 hours before assessment of PWT.

The results have demonstrated, as shown in Figure 2, that oral administration of Compound I of present invention significantly reduced mechanical allodynia in CCI rats of neuropathic pain model in a dose-dependent manner. In addition, at the same oral dose of 100 mg/kg, Compound 1 is about 100% more effective in suppressing mechanical allodynia in CCI neuropathic pain compared to gabapentin, the current gold standard medication for neuropathic pain. Of note, CCI-rats dosed with gabapentin have shown drowsiness or motor incoordination. which is consistent with known side effect of gabapentin. However, no such effect or other abnormality was observed in CCI-rats dosed with Compound I.

Spina! Nerve Ligation (SNL) Mono-Neuropathic Pais Model is Rat. The surgical procedure will be performed according to the method firstly described by Kim and Chung (Kim SH, Chung JM. Pain, 1992;50(3):355-63,). This procedure will result in tactile aliodynia in the left hindpaw. Rats will be included in the study only if they do not exhibit motor dysfunction (e.g., paw dragging or dropping) and their PWT is below to 4.0 g. The dose-response anti-allodvnia effects of test compound: on day 14 after surgery, rats were treated with test article at one of four doses, vehicle or positive control by oral gavage, and PWT was determined by calibrated von Frey filaments at time points of 0 (right before the drug dosing, Pre-Dose Baseline), 0.5, 1, 2, 4 and 6 hr. The a ti-allodynia effects of repeated administration of test article : Administration of test, article was started on day 7 after surgery, once a day for 7 days. PWT was determined by calibrated von Frey filaments, 2 hour after test article dosing each day. After 7 days dosing, the measurement were continued, every other day without compound dosing for another 7 days. PWT was determined at the time points as given above. The results have demonstrated that oral administration of Compound I of present invention significantly reduced mechanical aliodynia in SNL rats of neuropathic pain model in a dose-dependent manner.

Streptozotocin-Induced Diabetic Poly-Neuropathic Palo. Model in Rat. Diabetic peripheral neuropathy is a long-term complication of diabetes mellitus. Rats were received i.p. injections of streptozotocin (STZ, 50 mg/kg dissolved in citrate buffer at pH 4.5 immediately before the injection) to induce insulin-dependent diabetes mellitus and produce tactile aliodynia. One week later, blood glucose level was assayed, from samples taken from the tail vein, using standard test strips and colorimeter. Only animals with a blood glucose level >350 mg/dL were considered diabetic and included for the testing. Typical features of neuropathic pain (tactile aliodynia) were developed inhindpaws beginning around 2 to 3 weeks after STZ injection. After 4 weeks, a stable level of allodyma was reached. At this point, the rats with PWT below 4.5 g were enrolled for compound testing. The allodynic state was remain intact until the 8 ^ta week after STZ injection. All animals were observed daily and weighed regularly during the study period. This model of neuropathic pain mimics the symptoms of neuropathy in diabetic patients (Lynch J] , 3rd, et al Eur J Pharmacol. 1999;364(2-3): 141-6; Calcutt NA, J Neurol Sci. 2004;220(1-2): 137- 9). The dose-response anti-allodynia effects of test compound: On day 28 after STZ injection, rats were treated with test compound at one of four doses, or controls (vehicle and positive) by oral gavage, and PWT was determined by calibrated von Frey filaments at time points of 0 (right, before the drug dosing, Pre-Dose Baseline), 0.5, 1, 2, 4 and 6 hr. Tolerance effects: 6 days following the day 28 test, i.e. on day 34 after STZ injection, the same procedure on day 28 was repeated on day 34 with the same group of STZ-rats treated with the same (effective) dose as on day 28. The two results of anti-allodynia effects of test compound as measured on day 28 and on day 34 were compared to see if there was any tolerance effect of test compound in animals. The anti-allodynia effects of repeated administration of test compound: Administration (p.o.) of test compound was started on day 21 after STZ injection, once a day for 7 days. PWT was determined by calibrated von Frey filaments once a day, 1 hour after compound dosing. After 7 days dosing, the measurement was continued, every other day without compound dosing for another 7 days. PWT was determined at the time points as given above.

Pharmaceutical Compositions of the lavention

In one aspect, the present invention provides pharmaceutical compositions comprising one or more compounds of the present invention including the compound having stmctural Compound I.

The present pharmaceutical compositions contain a therapeutically effective amount of the present invention, preferably in purified form, together with a suitable amount of a pharmaceutically acceptable vehicle, so as to provide a form for proper administration to a patient. When administered to a patient, the present compounds and the pharmaceutically acceptable vehicles are preferably sterile. Water is a preferred vehicle when a compound is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions. Suitable

pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present pharmaceutical compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.

Pharmaceutical compositions may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries, which facilitate processing of compounds of the invention into preparations that can be used

pharmaceutically. Proper formulation is dependent, upon the route of administration chosen.

The present pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In some embodiments, the pharmaceutically acceptable vehicle is a capsule (see e.g., Grosswald et al, United States Patent No. 5,698,155). Other examples of suitable

pharmaceutical vehicles have been described in the art (see Remington: The Science and Practice of Pharmacy, Philadelphia College of Pharmacy and Science, 20 ^th Edition, 2000).

For topical administration the compound may be formulated as solutions, gels, ointments, creams, suspensions, etc. as is well-known in the art.

Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration. Systemic

formulations may be made in combination with a further active agent.

In some embodiments, the present compounds, are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compounds for intravenous administration are solutions in sterile isotonic aqueous buffer. For injection, the present compounds, or salts, solvates, esters, and/or prodrugs thereof, may be formulated in aqueous solutions, preferably, in physiologically compatible buffers such as Hanks' solution. Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. When necessary, the pharmaceutical compositions may also include a. solubilizing agent,

Pharmaceutical compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a.

lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or saehette indicating the quantity of active agent. When the present compounds, are administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When the present compounds are administered by injection, an ampoule of sterile water for injection or saline can be provided so that, the ingredients may be mixed prior to administration.

For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Pharmaceutical compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered pharmaceutical compositions may contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry coloring agents and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds of the invention. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time delay material such as glycerol monostearate or glycerol stearate may also be used. Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such vehicles are preferably of pharmaceutical grade.

For oral liquid preparations such as, for example, suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, saline, alkyienegiycols (e.g., propylene glycol), polyalkylene glycols (e.g., polyethylene glycol) oils, alcohols, slightly acidic buffers between pH 4 and pH 6 (e.g., acetate, citrate, ascorbate at between about 5.0 mM to about 50.0 niM) etc. Additionally, flavoring agents, preservatives, coloring agents, bile salts, aeylcarnitines and the like may be added.

For buccal a dministration, the pharmaceutical compositions may take the form of tablets, lozenges, etc. formulated in conventional manner.

liquid drug formulations suitable for use with nebulizers and liquid spray devices and

EHD aerosol devices will typically include a compound of the invention with a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutically acceptable vehicle is a liquid such as alcohol, water, polyethylene glycol or a perfluorocarbon. Optionally, another material may be added to alter the aerosol properties of the solution or suspension of compounds disclosed herein. Preferably, this material is liquid such as an alcohol, glycol, polyglycol or a fatty acid. Other methods of formulating liquid drug solutions or suspension suitable for use in aerosol devices are know n to those of skill in the art (see, e.g., Biesalski, United States Patent No. 5,1 12,598; Biesalski, United States Patent No. 5,556,61 1 ).

The present compounds may also be formulated in rectal or vaginal pharmaceutical compositions such as suppositories or retention enemas, e.g., containing conventional suppositor bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the present compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the present compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Therapeutic Doses

The present eompound(s), their crystalline form(s) and a. pharmaceutically acceptable vehicle is provided, will generally be used in an amount effective to treat or prevent diseases or disorders including: cancer, anxiety, generalized pain disorder, acute pain, chronic pain, inflammatory pain, and neuropathic pain.

^' The amount of the present compounds that will be effective in the treatment of a particular disorder or condition disclosed herein will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques known in the art. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The amount of the present compounds administered will, of course, be dependent on, among other factors, the subject being treated, the weight of the subject, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

For example, the dosage may be delivered in a pharmaceutical composition by a single administration, by multiple applications or controlled release. In some embodiment, the present compounds are delivered by oral sustained release administration. Dosing may be repeated intermittently, may be provided alone or in combination with other drags and may continue as long as required for effective treatment of the disease state or disorder.

Suitable dosage ranges for oral administration to a patient in need depend on the potency of the present compounds, but, are generally between about 0.1 mg to about 800 mg of a compound of the in vention per kilogram body weight; more preferably, between about 0.01 mg to about 50 mg of a compound of the invention per kilogram body weight; still more preferably, between about 0.05 mg to about 20 mg of a compound of the invention per kilogram body weight; and the patient is an animal; more preferably, a mammal; and most preferably, a human. Dosage ranges may be readily determined by methods known to the artisan of ordinary skill.

Suitable dosage ranges for intravenous (i.v.) administration to a patient in need are about 0.1 mg to about 200 mg per kilogram body weight; more preferably, between about 0.0.1 mg to about 20 mg of a compound of the invention per kilogram body weight; and the patient is an animal; more preferably, a mammal; and most preferably, a human.

Suitable dosage ranges for intranasal administration to a patient in need are generally about 0.1 mg/kg body weight to about 10 mg/kg body weight; more preferably, between about 0.01 mg to about 1 mg of a compound of the invention per kilogram body w ^r eight; and the patient is an animal; more preferably, a. mammal; and most preferably, a human.

Suppositories generally contain about 0.1 milligram to about 150 milligrams of a compound of the invention per kilogram body weight and comprise active ingredient in the range of about 0.5% to about 10% by weight.

Recommended dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual or intracerebral administration are in the range of about 0.1 mg to about 800 mg per kilogram of body weight; and the patient is an animal; more preferably, a mammal; and most preferably, a human. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Such animal models and systems are well-known in the art.

The present compounds are preferably assayed in vitro and in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays can be used to determine whether administration of a specific compound of the invention or a combination of compounds is preferred for reducing the pain or killing the cancer cells. The present compounds may also be demonstrated to be effective and safe using animal model systems.

Preferably, a therapeutically effective dose of the present compounds will provide therapeutic benefit without causing substantial toxicity. Toxicity of the present, compounds may be determined using standard pharmaceutical procedures and may be readily ascertained by the skilled artisan. The dose ratio between toxic and therapeutic effect is the therapeutic index. The present compounds generally exhibit particularly high therapeutic indices in treating associated disease and disorders or conditions. The dosage of the present compounds will preferably be within a range of circulating concentrations that include an effecti ve dose with little or no toxicity.

Combination Therapy

In certain embodiments of the present invention, the present compounds can be used in combination therapy with at least one additional active or therapeutic agent. The present compounds and the at least one additional active or therapeutic agent can act additively or, more preferably, synergistically. In some embodiments, the present compounds are administered concurrently, sequentially, or separately with the administration of another therapeutic agent. Exemplar}' active agents include, but are not limited to, aceglatone, aelarubiein, aitretamine, aminoglutethimide; 5-aminogieavulinic acid, amsacrine, anastrozole, ancitabine hydrochloride, 17- la antibody, antilymphocyte immunoglobulins, antineopiaston alO, asparaginase, pegaspargase, azacitidine, azathioprine, batimastai benzoporphyrin derivative, bicalutamide, bisantrene hydrochloride, bleomycin sulphate, brequinar sodium, broxuridine, busulphan, campath-ih, caracemide, carbetimer, carboplatin, carboquone, carmofur, carmustine, chlorambucil, chlorozotocin, chromomycin, cisplatin, cladribine, corynebacterium parvurn, cyclophosphamide, cyclosporin, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, diaziquone, dichlorodiethylsulphide, dideninin b,, docetaxel, doxifluridine, doxorubicin hychioride, droloxifene, echinomycin, edatrexate, elliptinium, elmustine, enloplatin, enocitabine, epirubicin hydrochloride, estramustine sodium phosphate, etanidazole, ethoglucid, etoposide, fadrozoie hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flutamide, formestane, fotemustine, gallium, nitrate, gencitabme, gusperimus, homoharringtonine, hydroxyurea, idarubicm hydrochloride, ifosfamide, ilmofosine, improsulfan tosylate, inolimomab, interleukin-2; irinotecan, jm-216, letrozole, lithium gamolenate, lobaplatin, lomustine, lonidamine, mafosfamide, meiphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, miboplatin, miltefosine, misonidazole, mitobronitol, mitoguazone dihydrochioride, mitolactol, mitomycin, mitotane, mitozanetrone hydrochloride, mizoribine, mopidamol, muitlaichilpeptide, muromonab-ecB , mustine

hydrochloride, mycophenolic acid, mycophenolate rnofedl, nedaplatin, nilutamide, nimustine hydrochloride, oxaliplatin, paclitaxel, perm, penostatin, peplomycin sulphate, pipobroman, pirarubicin, piritrexim isethionate, piroxantrone hydrochloride, plicamycin, porfimer sodium, prednirnustine, procarbazine hydrochloride, raltitrexed, ranimustine, razoxane, rogletimide, roquinimex, sebriplatin, semustine, sirolimus, sizofiran, sobuzoxane, sodium bromebrate, sparfosic acid, sparfosate sodium, sreptozocin, sulofenur, tacrolimus, tamoxifen, tegafur, teloxantrone hydrochloride, temozolomide, teniposide, testolactone, tetrasodium

mesotetraphenylporphine-sulphonate, thioguanine, thioinosine, thiotepa, topotecan, toremifene, treosulfan, trimetrexate, trofosfamide, tumor necrosis factor, ubenimex, uramustine, vinblastine sulphate, vincristine sulphate, vindesine sulphate, vinorelbine tartrate, vorozole, zinostatin, zolimomab aritox, zorubicin hydrochloride, an inhibitor of protein kinase A (P A) or PKC, an inhibitor of cAMP signaling, a nonsteroidal anti~infla.mnia.tory drag, a prostaglandin synthesis inhibitor, a local anesthetic, an anticonvulsant, an antidepressant, an opioid receptor agonist, and a neuroleptic, a benzodiazepine, a barbiturate, a neurosteroid and a inhalation anesthetic, a anesthetic and another pain killer and the like, either individually or in any combination.

All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood therefrom as modifications will be obvious to those skilled in the art. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description . The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein.

Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any

combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.