HK1109401A - Substituted heterocyclic compounds and uses thereof - Google Patents

Abstract

The present invention relates to substituted heterocyclic compounds and compositions comprising a substituted heterocyclic compound. The present invention also relates to methods for preventing or treating various diseases and disorders by administering to a subject in need thereof one or more substituted heterocyclic compounds. In particular, the invention relates to methods for preventing or treating cancer or an inflammatory disorder by administering to a subject in need thereof one or more substituted heterocyclic compounds. The present invention further relates to articles of manufacture and kits comprising one or more substituted heterocyclic compounds.

Description

Substituted heterocyclic compounds and uses thereof

This application claims priority to U.S. provisional application 60/607,408 filed on 3.9.2004, the entire contents of which are incorporated herein by reference.

1. Field of the invention

The present invention relates to substituted heterocyclic compounds and compositions comprising one or more of said compounds. The present invention also relates to methods of preventing or treating various diseases and disorders by administering one or more substituted heterocyclic compounds to a subject in need thereof. In particular, the present invention relates to methods of preventing or treating cancer or inflammatory disorders by administering one or more substituted heterocyclic compounds to a subject in need thereof.

2. Background of the invention

2.1 microtubes

The cytoskeleton of eukaryotic cells consists of a broad network of microfilaments, microtubules and intermediate filaments. Microtubules play an important role in mitosis. The α -, β -and γ -tubulin subunits are eukaryotic cytoskeletal proteins responsible for the formation of microtubules. Microtubules are hollow cylinders consisting of α -, β -tubulin heterodimers and connected end-to-end along the axis of the microtubule. Gamma-tubulin is contained in the microtubule tissue. Once formed, microtubules are in equilibrium and tubulin heterodimers are continually added to one end of the microtubules and removed at the opposite end. This balance allows the length of the microtubules to be controlled, and this control is necessary for the microtubules to perform their various functions in the cell.

During cell division, microtubules are responsible for transporting the set of daughter chromosomes to each individual daughter cell. Specifically, the DNA in the nucleus of the pre-mitotic nucleus is replicated and the two sets of genetic material are organized into separate sets of daughter chromosomes. Towards the end of the prophase division, microtubules grow from centrosomes at either end of the dividing parent cell towards two identical sets of chromosomes. This growing bundle of microtubules forms a structure called the mitotic spindle. The metaphase microtubules attach themselves to the chromosomes during the presortion, and once in the anaphase of division, the microtubules become unstable and shorten, separating the daughter chromosomes into their respective daughter cells at the opposite end of the dividing cell. Therefore, microtubules are intimately involved in the cell division process.

2.2 cancer and neoplastic diseases

Current Cancer therapies include surgery, chemotherapy and/or radiation therapy to eradicate tumor cells in a patient (see, e.g., Stockdale, 1998, "Principles of Cancer Patientmanagement" in Scientific American: Medicine, vol.3, Rubenstein and Federman, eds., Chapter 12, section IV)). All of these approaches have significant drawbacks for the patient. Surgery may be contraindicated, for example, due to the patient's health, or unacceptable to the patient. In addition, surgery may not completely eradicate the tumor tissue. Radiation therapy is effective only when irradiated tumor tissue exhibits higher radiation sensitivity than normal tissue, and radiation therapy often causes serious side effects. (same as the former)

With respect to chemotherapy, a number of chemotherapeutic agents may be used to treat neoplastic diseases. Specific examples of chemotherapeutic agents include agents that target tubulin (e.g., inhibit polymerization or stability of tubulin) or microtubules, such as colchicine (an alkaloid extracted from colchicine), vinca alkaloid agents (e.g., vincristine, vinblastine, and anhydrovinblastine (vinorelbine)), and taxane agents (e.g., paclitaxel (Taxol ®) and docetaxel (Taxotere ®)). Colchicine exerts its cytotoxic effect by binding to tubulin heterodimers through a single high affinity binding site (called the colchicine site). This binding initiates changes in the structure of the dimer and prevents the assembly of the dimer into microtubules. The colchicine binding site exhibits affinity for a diverse group of molecular structures including, but not limited to, podophyllotoxins (podophyllotoxins), eleutheropsins (stepanacin), chalcones (chalcons), noconazole (nocodazole), and TN-16. Exposure of rapidly dividing cells (e.g., cancer cells) to colchicine results in the disappearance of the mitotic spindle, blocking the cell in the M phase of the cell cycle and ultimately killing the cell. The binding of vinca alkaloids to a site on β -tubulin (referred to as the vinca alkaloid binding site) results in destabilization of the tubulin dimer. The poisoned dimer may then be added to the microtubule polymer and prevent further growth of the microtubules. Taxanes bind directly to the tubulin subunit of intact microtubules, stabilizing microtubules and inhibiting depolymerization or stability. When dividing cells enter anaphase, the stabilized microtubules cannot contract and cannot pull the sets of daughter chromosomes into their corresponding daughter cells. Thus, cell division cannot occur, and cells are blocked in the M phase of the cell cycle and eventually die.

Despite the availability Of a variety Of chemotherapeutic agents, conventional chemotherapy has a number Of drawbacks (see, e.g., Stockdale, 1998, "Principles Of Cancer Patient Management" in scientific American Medicine, vol.3, Rubenstein and Federman, eds., Chapter 12, section 10)). Almost all chemotherapeutic agents are toxic and can cause significant and often dangerous side effects, including severe nausea, bone marrow suppression, immunosuppression, and the like. In addition, many tumor cells are resistant or develop resistance to chemotherapeutic agents due to multiple drug resistance. Accordingly, there is a need in the art for novel compounds, compositions and methods for treating cancer or neoplastic disease with minimal or no side effects. In addition, there is a need for cancer treatments that provide cancer cell-specific therapies that specifically enhance the reduction in toxicity.

2.3 inflammatory diseases

Inflammation plays a major role in host defense and the progression of immune-mediated diseases. The initiation of the inflammatory response is due to a complex cascade of events, including chemical mediators (e.g., cytokines and prostaglandins) and inflammatory cells (e.g., leukocytes), in response to injury (e.g., trauma, ischemia, and foreign particles) and infection (e.g., bacterial or viral infection). The inflammatory response is characterized by increased blood flow, increased capillary permeability, and influx of phagocytes. These events result in the formation of swelling, congestion, increased temperature (altered temperature pattern) and pus at the site of injury or infection.

Cytokines and prostaglandins control the inflammatory response and are released into the blood or infected tissue through an ordered and self-limiting cascade. This release of cytokines and prostaglandins increases blood flow to the injured or infected area and can lead to congestion and increased temperature. Some of these chemicals cause fluid to penetrate into the tissue causing swelling. This protective process can stimulate nerves and cause pain. It is beneficial to the body if these changes persist for a limited time in the relevant area.

The finely balanced interactions between humoral and cellular immune elements in the inflammatory response may eliminate harmful substances and initiate repair of damaged tissues. When this finely balanced interaction is disrupted, the inflammatory response can cause considerable damage to normal tissue, which can be more dangerous than the original damage that initiated the response. In these uncontrolled inflammatory responses, clinical intervention is required to prevent tissue damage and organ dysfunction. Diseases such as rheumatoid arthritis, osteoarthritis, crohn's disease, asthma, allergy or inflammatory bowel disease are characterized by chronic inflammation.

Current treatments for inflammatory diseases include symptomatic drug therapy and immunosuppressants to control symptoms. For example, non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, fenoprofen, naproxen, tolmetin, sulindac, meclofenamate sodium, piroxicam, flurbiprofen, diclofenac, oxaprozin, nabumetone, etodolac, and ketoprofen have analgesic and anti-inflammatory effects. However, NSAIDs are not thought to alter disease progression. (Tierney et al (eds.), Current Medical diagnostics & Treatment, 37 th edition, Appleton & Lange (1998), p.793). In addition, NSAIDs often cause gastrointestinal side effects, affect the lower digestive tract causing perforation or aggravating inflammatory bowel disease, produce nephrotoxicity and prolonged bleeding time. Corticosteroids are another class of drugs commonly used to control inflammatory symptoms. Corticosteroids, like NSAIDs, do not alter the natural progression of the disease, so when the drug is discontinued, the clinical symptoms of active disease often reappear. Serious problems of adverse reactions caused by long-term corticosteroid treatment (e.g. osteoporosis, increased risk of infection, increased appetite, hypertension, edema, peptic ulcer, psychosis) greatly limit its long-term use.

Low doses of immunosuppressive agents (e.g., cytotoxic agents) are also commonly used to treat inflammatory diseases. Methotrexate (a folate antagonist) is frequently used, for example, in the treatment of psoriasis, rheumatoid arthritis and other inflammatory diseases. Like other cytotoxic agents, methotrexate often causes stomatitis, erythema, latecia, nausea, vomiting, diarrhea and damage to major organs (e.g., kidney and liver). Long-term use of immunosuppressive agents often leaves the patient unresponsive to infection.

New approaches to the treatment of inflammatory diseases are continually being sought. In particular, there is a constant search for any new therapy that reduces the dose and/or frequency of administration of currently used drugs, or that enables the currently used therapy to be more effective.

2.4 diseases of the Central nervous System

Central nervous system diseases affect a wide range of people with varying degrees of severity. Generally, one of the major features of this disease involves severe impairment of cognition or memory, manifested as a significant decline in the previous level of function. Dementia, for example, is characterized by several cognitive impairments, including severe memory deficits, and may be an independent or fundamental feature of various diseases, including alzheimer's disease, parkinson's disease, huntington's disease, and multiple sclerosis, among others. Other central nervous system disorders include delirium or disturbance of consciousness occurring for a short time, and amnesia or impaired discrete memory occurring without other central nervous system damage.

3. Summary of the invention

The present invention provides novel compounds, novel pharmaceutical compositions and the use of those compounds or pharmaceutical compositions in the prevention, treatment or management of various disorders. The invention provides, inter alia, methods for preventing, managing or treating cancer, including cancers that are refractory or unresponsive to conventional or currently available cancer treatments, comprising administering to a patient in need thereof an effective amount of a compound of the invention.

The present invention provides compounds having formula I and the compounds of table 1 below.

In one embodiment, the present invention provides a compound having formula I and pharmaceutically acceptable salts, solvates, or hydrates thereof, wherein X, R₁、R₂、R₃、R₄、R₅、R_aAnd R_bAs described herein.

The invention also provides pharmaceutical compositions comprising one or more compounds of the invention, or a pharmaceutically acceptable salt, solvate or hydrate thereof, and in particular pharmaceutical compositions comprising one or more compounds of the invention.

The invention provides pharmaceutical compositions comprising one or more compounds of the invention, or a pharmaceutically acceptable salt, solvate or hydrate thereof, and one or more prophylactic or therapeutic agents known to be useful, or already in use, or currently in use, for the prevention, treatment or amelioration of diseases or disorders associated with or characterized by abnormal angiogenesis, central nervous system disorders, proliferative disorders, inflammation, or diseases or disorders prevented, controlled, treated or ameliorated by inhibition of phosphodiesterase IV ("PDE 4") activity and/or inhibition of tubulin polymerization or stability, or a symptom or symptoms thereof. In another embodiment, a pharmaceutical composition of the invention may comprise one or more compounds of the invention, or a pharmaceutically acceptable salt, solvate or hydrate thereof, and one or more vascular targeting agents.

The invention also provides a method of inhibiting or reducing tubulin polymerization or stability, comprising contacting a cell with an effective amount of a compound of the invention.

The invention also provides a method of inhibiting or reducing tubulin polymerization or stability and PDE4 activity, comprising contacting a cell with an effective amount of a compound of the invention.

The invention also provides a method of inhibiting PDE4 activity comprising contacting a cell with an effective amount of a compound of the invention.

The invention also provides a method of targeting, blocking or disrupting vascular function in a tumor comprising contacting the tumor with an effective amount of a compound of the invention.

The invention also provides a method of targeting, blocking or destroying the vascular endothelium of a tumor comprising contacting the tumor with an effective amount of a compound of the invention.

The invention also provides a method of occluding existing tumor vessels comprising contacting a tumor with an effective amount of a compound of the invention.

The invention also provides a method of killing a tumor cell, comprising contacting a tumor cell with an effective amount of a compound of the invention.

The invention also provides a method of causing acute vascular collapse in a tumor cell, comprising contacting a tumor cell with an effective amount of a compound of the invention.

The invention also provides a method of blocking angiogenesis by vascular inhibition, the method comprising contacting a cell with an effective amount of a compound of the invention.

The present invention provides a method of inhibiting angiogenesis, said method comprising administering to a subject in need thereof an effective amount of a compound of the present invention.

In another embodiment, the invention provides a method of preventing, treating, managing or ameliorating a proliferative disorder or inflammation or one or more symptoms thereof, said method comprising administering a prophylactically or therapeutically effective amount of a compound of the invention alone or in combination with a prophylactically or therapeutically effective amount of one or more therapeutic agents other than a compound of the invention for preventing, treating, managing or ameliorating a proliferative disorder or inflammation or one or more symptoms thereof or known to be effective for preventing, treating, managing or ameliorating a proliferative disorder or inflammation or one or more symptoms thereof.

In another embodiment, the invention provides a method of preventing, treating, managing or ameliorating a central nervous system disorder or one or more symptoms thereof, which comprises administering a prophylactically or therapeutically effective amount of a compound of the invention alone or in combination with a prophylactically or therapeutically effective amount of one or more therapeutic agents other than a compound of the invention for preventing, treating, managing or ameliorating a central nervous system disorder or one or more symptoms thereof or known to be effective for preventing, treating, managing or ameliorating a central nervous system disorder or one or more symptoms thereof.

In a particular embodiment, the invention provides a method of preventing, treating, managing or ameliorating a tubulin-binding agent (e.g., colchicine, paclitaxel or vinca alkaloid) resistant cancer or one or more symptoms thereof, the method comprising administering a prophylactically or therapeutically effective amount of a compound of the invention alone or in combination with a prophylactically or therapeutically effective amount of one or more therapeutic agents other than a compound of the invention (e.g., colchicine, paclitaxel or vinca alkaloid) that are useful for preventing, treating, managing or ameliorating a proliferative disorder or inflammation or one or more symptoms thereof or that are known to be effective for preventing, treating, managing or ameliorating a proliferative disorder or inflammation or one or more symptoms thereof.

3.1 terms and abbreviations

The term "alkoxy" as used herein refers to compounds having the formula-O-alkyl, -O-lower alkyl, -O-cycloalkyl, -O-lower alkyl-cycloalkyl, -O-benzyl, -O-lower alkyl-benzyl, wherein alkyl, lower alkyl and cycloalkyl are defined below. Representative groups of-O-lower alkyl include, but are not limited to, -O-methyl, -O-ethyl, -O-n-propyl, -O-n-butyl, -O-n-pentyl, -O-n-hexyl, -O-n-heptyl, -O-n-octyl, -O-isopropyl, -O-sec-butyl, -O-isobutyl, -O-tert-butyl, -O-isopentyl, -O-2-methylbutyl, -O-2-methylpentyl, -O-3-methylpentyl, -O-2, 2-dimethylbutyl, -O-2, 3-dimethylbutyl, -O-2, 2-dimethylpentyl, -O-2, 3-dimethylpentyl, -O-3, 3-dimethylpentyl, -O-2, 3, 4-trimethylpentyl, -O-3-methylhexyl, -O-2, 2-dimethylhexyl, -O-2, 4-dimethylhexyl, -O-2, 5-dimethylhexyl, -O-3, 5-dimethylhexyl, -O-2, 4-dimethylpentyl, -O-2-methylheptyl, -O-3-methylheptyl, -O-vinyl, -O-allyl, -O-1-butenyl, -O-2-butenyl, O-2-pentyl, O-2-hexyl, O-2-pentyl, O, -O-isobutenyl, -O-1-pentenyl, -O-2-pentenyl, -O-3-methyl-1-butenyl, -O-2-methyl-2-butenyl, -O-2, 3-dimethyl-2-butenyl, -O-1-hexyl, -O-2-hexyl, -O-3-hexyl, -O-ethynyl, -O-propynyl, -O-1-butynyl, -O-2-butynyl, -O-1-pentynyl, -O-2-pentynyl, and-O-3-methyl-1-butynyl. Representative groups of-O-cycloalkyl include, but are not limited to, -O-cyclopropyl, -O-cyclobutyl, -O-cyclopentyl, -O-cyclohexyl, -O-cycloheptyl, -O-cyclooctyl, -O-cyclononyl, and-O-cyclodecyl. Representative of-O-lower alkyl-cycloalkyl groups include, but are not limited to, -O-CH ₂-cyclopropyl, -O-CH₂-cyclobutyl, -O-CH₂-cyclopentyl, -O-CH₂-cyclohexyl, -O-CH₂-cycloheptyl, -O-CH₂-cyclooctyl, -O-CH₂-cyclononyl, -O-CH₂-cyclodecyl, -O- (CH)₂)₂-cyclopropyl, -O- (CH)₂)₂-cyclobutyl, -O- (CH)₂)₂-cyclopentyl, -O- (CH)₂)₂-cyclohexyl, -O- (CH)₂)₂-cycloheptyl, -O- (CH)₂)₂-cyclooctyl, -O- (CH)₂)₂-cyclononyl and-O- (CH)₂)₂-a cyclodecyl group.

The term "alkyl" as used herein refers to a straight or branched chain saturated or unsaturated hydrocarbon having from 1 to 20 carbon atoms. Representative groups of straight chain alkyl groups include, but are not limited to-methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl, -n-decyl, -n-undecyl, -n-dodecyl, -n-tridecyl, -n-tetradecyl, n-pentadecyl, and the like; branched alkyl groups include, but are not limited to-isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl, 2-dimethylpentyl, 2, 3-dimethylpentyl, 3-dimethylpentyl, 2, 3, 4-trimethylpentyl, 3-methylhexyl, 2-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 3, 5-dimethylhexyl, 2, 4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl; unsaturated alkyl groups include, but are not limited to-vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2, 3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -ethynyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-butynyl, -1-hexynyl, -2-hexynyl, -1-heptynyl, -2-heptynyl, -1-octynyl, -2-octynyl, -1-nonynyl, -2-nonynyl, -1-decynyl, -2-decynyl.

The term "alkenyl" as used herein refers to a straight or branched chain acyclic hydrocarbon having from 2 to 10 carbon atoms and including at least one carbon-carbon double bond. Representative groups of straight and branched alkenyl groups include-vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2, 3-dimethyl-2-butenyl, -1-hexenyl, -2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl, -3-heptenyl, -1-octenyl, -2-octenyl, -3-octenyl, -1-nonenyl, -2-nonenyl, -3-nonenyl, -1-decenyl, -2-decenyl, -3-decenyl, and the like.

The term "alkynyl" as used herein refers to a straight or branched chain acyclic hydrocarbon having from 2 to 10 carbon atoms and including at least one carbon-carbon triple bond. Straight and branched chain- (C)₂-C₁₀) Representative groups of alkynyl include-ethynyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-butynyl, -4-pentynyl, -1-hexynyl, -2-hexynyl, -5-hexynyl, -1-heptynyl, -2-heptynyl, -6-heptynyl, -1-octynyl, -2-octynyl, -7-octynyl, -1-nonynyl, -2-nonynyl, -8-nonynyl, -1-decynyl, -2-decynyl, -9-decynyl, and the like. Alkynyl groups may be unsubstituted or substituted.

The term "antibody" as used herein refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized (camelized) antibodies, chimeric antibodies, single domain antibodies, single chain fv (scfv), single chain antibodies, Fab fragments, F (ab') fragments, disulfide linked fv (sdfv), and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-idiotypic antibodies against an antibody of the invention), and epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules, such as molecules that include an antigen binding site. The immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG)₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂) Or a subclass.

The term "aryl" as used herein refers to carbocyclic aryl groups. Examples of aryl groups include, but are not limited to, phenyl, benzyl, naphthyl, and anthracenyl.

The term "cycloalkyl" as used herein refers to a 3-, 4-, 5-, 6-, 7-, or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative of C₃-C₈Cycloalkyl groups include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1, 3-cyclohexadienyl, -1, 4-cyclohexadienyl, -cycloheptyl, -1, 3-cycloheptadienyl, -1, 3, 5-cycloheptatrienyl, cyclooctyl and cyclooctadieneyl . The term "cycloalkyl" also includes-lower alkyl-cycloalkyl, wherein lower alkyl and cycloalkyl are as defined herein. Examples of-lower alkyl-cycloalkyl include, but are not limited to, -CH₂-cyclopropyl, -CH₂-cyclobutyl, -CH₂-cyclopentyl, -CH₂-cyclopentadienyl, -CH₂-cyclohexyl, -CH₂-cycloheptyl and-CH₂-a cyclooctyl group.

The terms "compound" and "compound of the invention" are used interchangeably herein and refer to any compound specifically described or generally disclosed herein, including pharmaceutically acceptable salts, hydrates, or solvates thereof. In one embodiment, the compounds of the present invention are compounds having formula I and the compounds in table 1, and pharmaceutically acceptable salts, hydrates, or solvates thereof.

As used herein, the terms "disorder" and "disease" are used interchangeably to refer to the discomfort of an individual. Certain maladies may be characterized by more than one condition. For example, certain discomforts may be characterized by both a non-cancerous proliferative disorder and an inflammatory disorder. In one embodiment, the proliferative disorder is cancer.

The term "effective amount" as used herein refers to an amount of a compound of the invention sufficient to reduce or ameliorate the severity or duration of a disorder (e.g., a disorder characterized by aberrant angiogenesis, a central nervous system disorder, a proliferative disorder, or a disorder characterized by inflammation (i.e., an inflammatory disorder)) or one or more symptoms thereof, prevent the development of a disorder (e.g., a disorder characterized by aberrant angiogenesis, a proliferative disorder, or an inflammatory disorder), cause regression of a disorder (e.g., a proliferative disorder or an inflammatory disorder), prevent the recurrence, development, or onset of one or more symptoms associated with a disorder (e.g., a disorder characterized by aberrant angiogenesis, a proliferative disorder, or an inflammatory disorder), or enhance or improve the prophylactic or therapeutic effect of another therapy. In a particular embodiment, an effective amount for cancer treatment refers to an amount of a compound of the invention that inhibits or slows the proliferation of cancer cells, inhibits or slows the spread (metastasis) of tumor cells, inhibits or slows the onset, development, or progression of one or more symptoms associated with cancer, reduces the size of a tumor, or kills tumor cells. In one embodiment, a therapeutically effective amount of a compound of the invention is an amount that attacks the tumor vasculature and shuts off the blood and/or oxygen supply to the tumor. Preferably, a therapeutically effective amount of a compound of the invention inhibits or reduces the proliferation of cancer cells or the size of a tumor by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as calculated relative to a control or placebo (e.g., phosphate buffered saline ("PBS"). In another embodiment, an effective amount with respect to inflammation refers to an amount of a compound of the invention that reduces inflammation of a joint, organ, or tissue. Preferably, an effective amount of a compound of the invention reduces inflammation of a joint, organ or tissue by at least 5%, preferably by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, calculated relative to a control or placebo (e.g., phosphate buffered saline). In another embodiment, an effective amount refers to an amount of a compound of the invention that reduces the Psoriasis Area and Severity Index (PASI) score of a human by at least 20%, at least 35%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%. In an alternative embodiment, an effective amount for the treatment of psoriasis preferably refers to an amount of a compound of the invention that increases the overall human rating score by at least 25%, at least 35%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. Examples of therapeutically effective amounts of the compounds of the invention are given in section 4.4.5, below.

The term "halogen" as used herein refers to-F, -Cl, -Br or-I.

The term "heterocycle" as used herein refers to an aromatic or non-aromatic cycloalkyl group in which 1 to 4 ring carbon atoms are independently replaced by a heteroatom selected from O, S and N. Representative examples of heterocycles include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolyl, pyrrolyl, pyrrolidinyl, thienyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, (1, 4) -dioxane, (1, 3) -dioxolane, 4, 5-dihydro-1H-imidazolyl, and tetrazolyl. The heterocyclic ring may be substituted or unsubstituted. The heterocyclic ring may also be bonded at any ring atom (i.e., at any carbon atom or heteroatom of the heterocyclic ring).

The term "in combination" as used herein refers to the use of more than one treatment (e.g., one or more prophylactic and/or therapeutic agents). The use of the term "in combination" does not limit the order in which treatment (e.g., prophylactic and/or therapeutic agents) is administered to an individual having a disorder (e.g., a disorder characterized by abnormal angiogenesis, a proliferative disorder, or an inflammatory disorder). A first treatment (e.g., a prophylactic or therapeutic agent, such as a compound of the invention) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a disorder (e.g., a disorder characterized by abnormal angiogenesis), Proliferative disorder or inflammatory disorder).

The term "isolated" as it appears with respect to a compound (e.g., a compound of the invention) refers to a compound that is substantially free of chemical precursors, other compounds (as chemically synthesized), or other isomers. In a particular embodiment, the compound is 60%, 65%, 75%, 80%, 85%, 90%, 95%, or 99% free of other different compounds (e.g., other isomers). Preferably the compounds of the invention are isolated compounds.

The term "lower alkyl" as used herein refers to a straight or branched chain saturated or unsaturated hydrocarbon having from 1 to 8 carbon atoms. Representative groups of straight chain lower alkyl include, but are not limited to-methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, and-n-octyl; branched lower alkyl includes, but is not limited to-isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, -2-methylbutyl, -2-methylpentyl, -3-methylpentyl, -2, 2-dimethylbutyl, -2, 3-dimethylbutyl, -2, 2-dimethylpentyl, -2, 3-dimethylpentyl, -3, 3-dimethylpentyl, -2, 3, 4-trimethylpentyl, -3-methylhexyl, -2, 2-dimethylhexyl, -2, 4-dimethylhexyl, -2, 5-dimethylhexyl, -3, 5-dimethylhexyl, -2, 4-dimethylpentyl, and, -2-methylheptyl, -3-methylheptyl. Unsaturated C ₁-C₈Alkyl groups include, but are not limited to-vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2, 3-dimethyl-2-butenyl, -1-hexyl, -2-hexyl, -3-hexyl, -ethynyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and-3-methyl-1-butynyl.

The term "lower hydroxyalkyl" as used herein refers to a lower alkyl group as described herein substituted with one or more hydroxy groups. Representative lower hydroxyalkyl groups include, but are not limited to, -CH₂OH、-(CH₂)₂OH、-(CH₂)₃OH、-(CH₂)₄OH、-(CH₂)₅OH、-CH(OH)CH₃、-CH(OH)CH₂CH₃、-CH(OH)(CH₂)₂CH₃、-CH₂CH(OH)CH₃、-CH₂CH(OH)CH₂CH₃And the like.

When the groups described herein are referred to as "substituted or unsubstituted," in the case of substitution, they may be substituted with any substituent that does not adversely affect the desired activity of the compound. Examples of preferred substituents are those of the exemplary compounds and compounds of the embodiments disclosed herein, as well as halogen (e.g., chlorine, iodine, bromine, or fluorine), C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, hydroxy, C_1-6Alkoxy, amino, nitro, mercapto, thioether, imine, cyano, amido, phosphonato, phosphine, carboxyl, thiocarbonyl, sulfonyl, sulfonamide, ketone, aldehyde, ester, acetyl, acetoxy, carbamoyl, oxo (═ O), haloalkyl (e.g. trifluoromethyl), substituted aminoacyl and aminoalkyl, carbocyclic cycloalkyl (which may be monocyclic or fused or non-fused polycyclic (e.g. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl)) or heterocycloalkyl (which may be monocyclic or fused or non-fused polycyclic (e.g. pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl or thiazinyl)), carbocyclic or heterocyclic, monocyclic or fused or non-fused polycyclic aryl (e.g. phenyl, naphthyl, pyrrolyl, indolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl), triazolyl, heteroaryl, carboxyl, thiocarbonyl, sulfonyl, sulfonamide, sulfonyl, ketone, aldehyde, ester, acetyl, carbamoyl, cyclopentyl, Tetrazolyl, pyrazolyl, pyridyl, quinolyl, isoquinolyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothienyl or benzofuranyl), amino (primary, secondary or tertiary), O-lower alkyl, O-aryl, aryl-lower alkyl, CO ₂CH₃、CONH₂、OCH₂CONH₂、NH₂、SO₂NH₂、OCHF₂、CF₃、OCF₃And may also optionally be fused ring structures or bridges (e.g. -OCH)₂O-or-O-lower alkyl-O-). These substituents may optionally be further substituted with substituents selected from such groups. In one embodiment, when lower alkyl (e.g., methylene) is substituted, it is substituted withSide chain substitution of natural amino acids.

Various compounds of the invention include one or more chiral centers and may exist as racemic mixtures of enantiomers, diastereomeric mixtures, or enantiomeric or optically pure compounds. The invention includes the use of stereoisomerically pure forms of these compounds, as well as the use of mixtures of those forms. For example, mixtures comprising equal or unequal amounts of the enantiomers of a particular compound of the invention may be used in the methods and compositions of the invention. These isomers can be asymmetrically synthesized or resolved by standard techniques, such as chiral columns or chiral resolving agents. See, e.g., Tetrahedron 33 by Enantiomers, Racemates and solutions, Jacques, J. et al (Wiley-Interscience, New York, 1981), Wilen, S.H. et al: 2725(1977), Stereochemistfy of carbon Compounds (McGraw-Hill, NY, 1962), Eliel, E.L., et al, and Tables of resolution Agents and Optical resolution, Wilen, S.H., pp.268 (E.L.Eliel eds., Univ.of Notre Dame Press, Notre Dame, IN, 1972).

It should also be noted that the compounds of the present invention include the E and Z isomers or mixtures thereof, as well as the cis and trans isomers or mixtures thereof. In certain embodiments, the compounds of the present invention are isolated as the E or Z isomers. In other embodiments, the compounds of the invention are mixtures of E and Z isomers.

As used herein, unless otherwise indicated, the term "stereomerically pure" refers to a composition that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of the compound, or a composition that comprises one geometric isomer of a compound (e.g., around a double bond) and is substantially free of other geometric isomers. For example, a stereoisomerically pure compound of the present invention having one chiral center or a composition thereof is substantially free of the opposite enantiomer of the compound. Stereoisomerically pure compounds of the present invention having two chiral centers, or compositions thereof, are substantially free of other diastereomers of the compound. Stereoisomerically pure compounds of the present invention having one double bond capable of forming the E/Z isomer or compositions thereof are substantially free of one of the E/Z isomers. A typical stereoisomerically pure compound comprises greater than about 80% by weight of one stereoisomer or E/Z isomer of the compound and less than about 20% by weight of the other stereoisomer or E/Z isomer of the compound, more preferably greater than about 90% by weight of one stereoisomer or E/Z isomer of the compound and less than about 10% by weight of the other stereoisomers or E/Z isomers of the compound, even more preferably greater than about 95% by weight of one stereoisomer or E/Z isomer of the compound and less than about 5% by weight of the other stereoisomers or E/Z isomers of the compound, most preferably greater than about 97% by weight of one stereoisomer or E/Z isomer of the compound and less than about 3% by weight of the other stereoisomer or E/Z isomer of the compound. As used herein, unless otherwise indicated, the term "stereoisomerically enriched" means that a compound of the present invention or a composition thereof comprises greater than about 60% by weight of one stereoisomer or E/Z isomer of a compound of the present invention, preferably greater than about 70% by weight, more preferably greater than about 80% by weight of one stereoisomer or E/Z isomer of a compound of the present invention. The term "enantiomerically pure" as used herein, unless otherwise specified, refers to a stereoisomerically pure compound of the invention or a composition thereof having one chiral center. Similarly, the term "stereoisomerically enriched" refers to a stereoisomerically enriched compound of the present invention or a composition thereof having one chiral center.

It should be noted that if the stereochemistry of a structure or a partial structure is not indicated with, for example, bold or dashed lines, the structure or partial structure is to be understood as encompassing all stereoisomers of it.

The term "control" as used herein refers to a beneficial result that an individual obtains from treatment (e.g., a prophylactic or therapeutic agent), but the beneficial result does not result in recovery from the disease. In certain embodiments, one or more treatments (e.g., one or more prophylactic or therapeutic agents) are administered to an individual to "manage" a disease or a symptom thereof and thereby prevent progression or worsening of the disease or symptom thereof.

The terms "non-responsive" and "refractory" as used herein describe individuals who are treated for a disorder (e.g., a disorder characterized by abnormal angiogenesis, a proliferative disorder, or an inflammatory disorder) with currently available treatments (e.g., prophylactic or therapeutic agents) that are not clinically adequate to alleviate one or more symptoms associated with such a disorder. Often such individuals suffer from severe persistent active disease and require additional treatment to ameliorate symptoms accompanying their disorder (e.g., a disorder characterized by abnormal angiogenesis, a proliferative disorder, or an inflammatory disorder).

The phrase "pharmaceutically acceptable salt" as used herein refers to pharmaceutically acceptable organic or inorganic salts of the compounds of the present invention. Preferred salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate (isonicotinate), lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, gluconate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1' -methylene-bis- (2-hydroxy-3-naphthoate)). The pharmaceutically acceptable salt may comprise another molecule, such as an acetate, succinate, or other counterion. The counter ion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Where the pharmaceutically acceptable salt has multiple charged atomic moieties, there can be multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterions.

The term "pharmaceutically acceptable solvate" as used herein refers to the association of one or more solvent molecules with a compound of the invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

The term "pharmaceutically acceptable hydrate" as used herein means that the compound of the present invention or a salt thereof further comprises a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

The term "preventing" as used herein refers to administering a treatment (e.g., a prophylactic or therapeutic agent) or a combination treatment (e.g., a prophylactic or therapeutic agent combination) to an individual to prevent the recurrence, onset, or progression of one or more symptoms of the disorder or condition from which it is suffering.

The phrase "prophylactically effective amount" as used herein refers to an amount of a treatment (e.g., a prophylactic agent) sufficient to prevent the development, recurrence, or onset of a disorder (e.g., a disorder characterized by aberrant angiogenesis, a proliferative disorder, or an inflammatory disorder) or one or more symptoms of a disorder, or to enhance or ameliorate the prophylactic effect of another treatment (e.g., another therapeutic agent). Examples of prophylactically effective amounts of the compounds are described in section 4.4.5, below.

The phrase "side effects" as used herein includes both undesirable and adverse effects of treatment (e.g., prophylactic or therapeutic agents). Side effects are always undesirable, but undesirable side effects are not necessarily undesirable. Adverse effects of treatment (e.g., prophylactic or therapeutic formulations) can be harmful or uncomfortable or dangerous. Side effects include, but are not limited to, fever, chills, lethargy, gastrointestinal toxicity (including ulcers and erosions of the stomach and intestines), nausea, vomiting, neurotoxicity, toxic renal damage, renal toxicity (including cases of papillary necrosis and chronic interstitial nephritis), hepatotoxicity (including elevated serum liver enzyme levels), bone marrow toxicity (including leukopenia, myelosuppression, thrombocytopenia and anemia), dry mouth, metallic taste, prolonged pregnancy, weakness, narcolepsy, pain (including muscle pain, bone pain and headache), hair loss, weakness, dizziness, extrapyramidal symptoms, akathisia, cardiovascular disorders and sexual dysfunction.

The terms "individual" and "patient" as used herein are used interchangeably herein. The term "individual" refers to an animal, preferably a mammal including a non-primate (e.g., cows, pigs, horses, cats, dogs, mice and mice) and a primate (e.g., apes such as macaques, chimpanzees and humans), more preferably a human. In one embodiment, a disorder (e.g., a disorder characterized by abnormal angiogenesis, a proliferative disorder, or an inflammatory disorder) in an individual is refractory or unresponsive to current treatment. In another embodiment, the subject is a domestic animal (e.g., a horse, cow, or pig) or a pet (e.g., a dog or cat). In another embodiment, the subject is not an immunocompromised or immunosuppressed mammal, preferably a human (e.g., an HIV subject). In another embodiment, the subject is not at a lymphocyte count of less than about 500 cells/mm ³Preferably a human. In a preferred embodiment, the individual is a human.

The term "synergistic" as used herein refers to a combination of a compound of the invention and another treatment (e.g., a prophylactic or therapeutic agent) that has been or is currently being used to prevent, control or treat a disorder (e.g., a disorder characterized by aberrant angiogenesis, a proliferative disorder or an inflammatory disorder), the combination being more effective than the additive effects of the treatments. The synergistic effect of the combination therapy (e.g., prophylactic or therapeutic agents in combination) allows for the use of a lower dose of one or more therapies and/or administration of treatments at a lower frequency to individuals suffering from a disorder (e.g., a disorder characterized by abnormal angiogenesis, a proliferative disorder, or an inflammatory disorder). The ability to use a lower dose of treatment (e.g., a prophylactic or therapeutic agent) and/or to administer such treatment less frequently reduces the toxicity associated with such treatment administered to a subject without reducing the effectiveness of such treatment in preventing, managing or treating a condition (e.g., a condition characterized by aberrant angiogenesis, a proliferative condition, or an inflammatory condition). In addition, the synergistic effect may improve the efficacy of the formulation in preventing, controlling or treating a disorder (e.g., a disorder characterized by abnormal angiogenesis, a proliferative disorder or an inflammatory disorder). The synergistic effect of the combination therapies (e.g., prophylactic or therapeutic agents in combination) can avoid or mitigate the adverse or undesirable side effects associated with the use of each therapy alone. In one embodiment, the term synergistic refers to the biological effect of a single compound of the invention on a tumor or tumor cells. Without being limited by theory, since the compounds of the present invention possess both vascular targeting activity (particularly effective against central tumor cells) and anti-angiogenic activity (particularly effective against peripheral tumor cells), the compounds of the present invention are particularly useful for eradicating a substantial portion of tumors, and in one embodiment, completely eradicate tumors. Therefore, the compounds of the present invention are able to very actively combat tumors due to the synergistic effect of their dual activities (vascular targeting agent and anti-angiogenic agent).

The term "treatment" as used herein refers to the administration of one or more treatments (e.g., one or more therapeutic agents such as a compound of the invention) to reduce or ameliorate the progression, severity and/or duration of a disorder (e.g., a disorder characterized by abnormal angiogenesis, a proliferative disorder or an inflammatory disorder), or to ameliorate one or more symptoms of a disorder. In a particular embodiment, such terms refer to inhibiting or slowing the proliferation of cancer cells, inhibiting or slowing the dissemination (metastasis) of tumor cells, inhibiting or slowing the onset, development or progression of one or more symptoms associated with cancer, reducing the size of a tumor, or improving the ECOG or Karnofsky score of a patient. In other embodiments, such terms refer to reducing swelling of one or more joints, organs, or tissues, or reducing pain associated with inflammatory conditions. In other embodiments, such terms refer to decreasing a human PASI score or improving a human overall rating score.

The term "tubulin binding agent", "tubulin binding agent" or variants thereof as used herein refers to any cell (proliferation) inhibitor or cytotoxic agent that can bind tubulin, alpha, beta-tubulin dimer or intact microtubules in a cell. In one embodiment, the tubulin binding agent inhibits the polymerization or stability of tubulin. In another embodiment, the tubulin binding agent is a tubulin destabilizing agent.

The term "inhibiting the polymerization or stability of tubulin" or "inhibition of the polymerization or stability of tubulin" as used herein refers to any change in the structure of tubulin dimer, any hindrance to the assembly of tubulin dimer into microtubules, or any destabilization of tubulin.

The following abbreviations are used herein and have the indicated definitions: Dess-Martin Periodinane is 1, 1, 1-triacetoxy-1, 1-dihydro-1, 2-benziodoxoyl-3- (1H) -one, DMF is N, N-dimethylformamide, DMSO is dimethyl sulfoxide, EtOAc is ethyl acetate, HPLC is high performance liquid chromatography, HUVEC is human umbilical vein endothelial cells, KHMDS is potassium hexamethyldisilazide, LHMDS is lithium hexamethyldisilazide, PBMC is peripheral blood mononuclear cells, PCC is pyridinium chlorochromate, PDC is pyridinium dichromate, Ph is phenyl, THF is tetrahydrofuran, TLC is thin layer chromatography, TPAP is tetra-N-propylamine perruthenate.

4. Detailed description of the invention

The invention provides compounds and uses of the compounds. The invention includes the use of a compound of the invention to inhibit the polymerization of tubulin and/or the stability of tubulin and/or to inhibit mitosis. The invention also includes the use of a compound of the invention to inhibit angiogenesis. The invention also includes the use of a compound of the invention to inhibit PDE4 activity. The invention also includes the use of the compounds of the invention as vascular targeting agents.

The invention encompasses treatment regimens that provide better prophylactic or therapeutic effects on multiple disorders (e.g., disorders characterized by aberrant angiogenesis, proliferative disorders, or inflammatory disorders) or one or more symptoms thereof than current single agent or combination therapies. In particular, the present invention provides prophylactic and therapeutic regimens for preventing, treating, managing or ameliorating a proliferative disorder (e.g., cancer), macular degeneration or an inflammatory disease, or one or more symptoms thereof, which comprise administering to a subject a prophylactically or therapeutically effective amount of one or more compounds of the invention, alone or in combination with a prophylactically or therapeutically effective amount of at least one other prophylactic or therapeutic agent other than a compound of the invention.

4.1 Compounds of the invention

The present invention includes compounds having formula I and the compounds listed in table 1 below.

In one embodiment, the present invention provides a compound having formula I and pharmaceutically acceptable salts, solvates, or hydrates thereof,

wherein:

x is a substituted or unsubstituted imidazole, a substituted or unsubstituted pyridine, a substituted or unsubstituted pyrrolidine, a substituted or unsubstituted thiophene, a substituted or unsubstituted indole, a substituted or unsubstituted 2, 3-dihydrobenzofuran, a substituted or unsubstituted 3, 4-dihydro-2H-benzo (b) (1, 4) oxazine, a substituted or unsubstituted 1H-benzo (d) (1, 2, 3) triazole, a substituted or unsubstituted quinoline, a substituted or unsubstituted benzofuran, a substituted or unsubstituted benzo (d) oxazol-2 (3H) one, or a substituted or unsubstituted pyrimidine;

R₁And R₂Each independently is-H, -CN, halogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -NHC (O) R₉、-NHC(O)OR₉-COOH, -C (O) -lower alkyl, -C (O) O-lower alkyl, -C (O) -N (R)₉)₂Substituted or unsubstituted aryl, or substituted or unsubstituted heterocycle;

R_aand R_bEach independently is-H, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, halogen, cyano, -NO₂、-OH、-OPO(OH)₂、-N(R₉)₂、-OC(O)-R₁₀、-OC(O)-R₁₀-N(R₁₀)₂、-C(O)N(R₁₀)₂、-NHC(O)-R₁₀、-NHS(O)₂-R₁₀、-S(O)₂-R₁₀、-S(O)₂-NH₂、-S(O)₂-N(R₁₀)₂、-NHC(O)NH-R₁₀、-NHC(O)N(R₁₀)₂、-NHC(O)NHSO₂-R₁₀、-NHC(O)-R₁₀-N(R₁₀)₂、-NHC(O)CH(R₁₀)(N(R₉)₂) or-NHC (O) -R₁₀-NH₂；

R₃is-H, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, halogen, cyano, -NO₂、-OH、-OPO(OH)₂、-N(R₉)₂、-OC(O)-R₁₀、-OC(O)-R₁₀-N(R₁₀)₂、-OC(O)-R₁₀-NH₂、-C(O)N(R₁₀)₂、-NHC(O)-R₁₀、-NHS(O)₂-R₁₀、-S(O)₂-R₁₀、-OS(O)₂-R₁₀、-S(O)₂-NH₂、-S(O)₂-N(R₁₀)₂、-OS(O)₂-NH₂、-OS(O)₂-N(R₁₀)₂、-NHC(O)O-R₁₀、-NHC(O)NH-R₁₀、-NHC(O)N(R₁₀)₂、-NHC(O)NHSO₂-R₁₀、-NHC(O)-R₁₀-N(R₁₀)₂、-NHC(O)CH(R₁₀)(N(R₉)₂) or-NHC (O) -R₁₀-NH₂Or R₃And R_aOr R₄Together form-O-C (R)₁₆R₁₇)O-、-O-(C(R₁₆R₁₇))₂-O-or-O- (C (R)₁₆R₁₇))₃-O-；

R₄is-H, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, substituted or unsubstitutedSubstituted alkoxy, halogen, cyano, -NO₂、-OH、-OPO(OH)₂、-N(R₉)₂、-OC(O)-R₁₀、-OC(O)-R₁₀-N(R₁₀)₂、-OC(O)-R₁₀-NH₂、-C(O)N(R₁₀)₂、-NHC(O)-R₁₀、-NHS(O)₂-R₁₀、-S(O)₂-R₁₀、-OS(O)₂-R₁₀、-S(O)₂-NH₂、-S(O)₂-N(R₁₀)₂、-OS(O)₂-NH₂、-OS(O)₂-N(R₁₀)₂、-NHC(O)O-R₁₀、-NHC(O)NH-R₁₀、-NHC(O)N(R₁₀)₂、-NHC(O)NHSO₂-R₁₀、-NHC(O)-R₁₀-N(R₁₀)₂、-NHC(O)CH(R₁₀)(N(R₉)₂) or-NHC (O) -R₁₀-NH₂；

R₅is-H, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, halogen, cyano, -NO ₂、-OH、-OPO(OH)₂、-N(R₉)₂、-OC(O)-R₁₀、-OC(O)-R₁₀-N(R₁₀)₂、-OC(O)-R₁₀-NH₂、-C(O)N(R₁₀)₂、-NHC(O)-R₁₀、-NHS(O)₂-R₁₀、-S(O)₂-R₁₀、-OS(O)₂-R₁₀、-S(O)₂-NH₂、-S(O)₂-N(R₁₀)₂、-OS(O)₂-NH₂、-OS(O)₂-N(R₁₀)₂、-NHC(O)O-R₁₀、-NHC(O)NH-R₁₀、-NHC(O)N(R₁₀)₂、-NHC(O)NHSO₂-R₁₀、-NHC(O)-R₁₀-N(R₁₀)₂、-NHC(O)CH(R₁₀)(N(R₉)₂) or-NHC (O) -R₁₀-NH₂；

Each R₉Each independently is-H, substituted or notSubstituted lower alkyl, or substituted or unsubstituted cycloalkyl;

each R₁₀Each independently is substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted lower hydroxyalkyl, or R₁₀And the nitrogen atom to which it is attached form a substituted or unsubstituted heterocyclic ring, or, where appropriate, R₁₀is-H; and

R₁₆and R₁₇Each independently is-H or halogen.

In another embodiment, the compounds having formula I are the following, wherein: (1) x is pyridine, substituted pyridine, pyrrolidine, imidazole, naphthalene or thiophene; (2) r_aAnd R_bIs H; and (3) R₄Hydrogen, nitro, cyano, trifluoromethyl, ethoxycarbonyl, methoxycarbonyl, propoxycarbonyl, acetyl, carbamoyl, acetoxy, carboxyl, hydroxyl, amino, lower alkyl, lower alkylenemethyl, lower alkoxy, or halogen; if R is₃Or R₅One is H, then the other is not-O-C_1-10Alkyl, -O-C_1-10Monocycloalkyl, -O-C_1-10Polycyclic alkyl radicals, -O-C_1-10Benzocycloalkyl, -C_0-3-C_1-10Alkyl, -C_0-3-C_1-10Monocycloalkyl, -C_0-3-C_1-10Polycyclic alkyl radical, -C_0-3-C_1-10Benzocycloalkyl, -CH ═ C _1-10Alkyl, -CH ═ C_1-10Monocyclic alkyl or-CH ═ C_1-10A bicycloalkyl group.

In another embodiment, the compounds having formula I are those wherein when X is substituted or unsubstituted imidazole, substituted or unsubstituted pyridine, substituted or unsubstituted pyrrolidine, or substituted or unsubstituted thiophene, if R is₃Or R₅One is H and the other is not substituted or unsubstituted alkyl, substituted alkenyl, or substituted or unsubstituted alkoxy.

In one embodiment, the compound having formula I is the followingWherein R is₁、R₃、R₄、R₅、R_aAnd R_bAs described above, X is a substituted or unsubstituted indole, a substituted or unsubstituted 2, 3-dihydrobenzofuran, a substituted or unsubstituted 3, 4-dihydro-2H-benzo (b) (1, 4) oxazine, a substituted or unsubstituted 1H-benzo (d) (1, 2, 3) triazole, a substituted or unsubstituted quinoline, a substituted or unsubstituted benzofuran, a substituted or unsubstituted benzo (d) oxazol-2 (3H) one, or a substituted or unsubstituted pyrimidine.

In another embodiment, the compound having formula I is wherein R is₁And R₂Those in which one is-H.

In another embodiment, the compound having formula I is wherein R is₃Or R₅Those which are alkoxy, preferably methoxy or ethoxy.

In another embodiment, the compound having formula I is wherein R is₃And R₅Those which are alkoxy, preferably methoxy or ethoxy.

In another embodiment, the compound having formula I is wherein R is₄And R₃Or R₅Those in which one of them is an alkoxy group, preferably a methoxy or ethoxy group.

In another embodiment, the compound having formula I is wherein R is₁Those compounds which are cyano.

In another embodiment, the compounds having formula I are those wherein X is substituted.

In another embodiment, the compounds having formula I are those wherein X is substituted with lower alkyl, preferably methyl.

Illustrative examples of the compounds of the present invention include the compounds listed in table 1 and pharmaceutically acceptable salts, solvates, or hydrates thereof. It should be noted that the E/Z and cis/trans isomers of these compounds are particularly contemplated.

TABLE 1

In particular, the invention includes compounds that inhibit or reduce tubulin polymerization and/or stability. In a particular embodiment, the invention includes compounds that inhibit or reduce tubulin polymerization or stability and inhibit or reduce the expression of one or more tumor necrosis factor-alpha (TNF-alpha) activities. In another embodiment, the invention encompasses compounds that inhibit or reduce tubulin polymerization or stability and inhibit or reduce the expression of one or more PDE4 activities. In another embodiment, the invention includes compounds having formula I that inhibit or reduce tubulin polymerization or stability, inhibit or reduce the expression of one or more TNF- α activities, and inhibit or reduce the expression of one or more PDE4 activities. In another embodiment, the invention comprises arresting the cell cycle in G ₂Compound of the/M stage.

As noted above, certain compounds of the present invention may include one or more chiral atoms. Accordingly, the present invention includes all stereoisomers (i.e., geometric isomers) including conformational and configurational isomers (e.g., enantiomers, diastereomers and mixtures thereof). In one embodiment, the invention includes the racemic or R-or S-enantiomers of all compounds described herein. Each enantiomer may be provided substantially free of the other enantiomer (e.g., at least 75% (w/w), at least 90% (w/w), or at least 99% (w/w) free of the other enantiomer) or as a mixture (e.g., a racemic mixture).

The compounds of the present invention also include alkenes, which if asymmetrically substituted may exist in the E and Z or cis and trans configurations. Thus, the present invention includes E and Z as well as cis and trans olefin isomers. For example, compounds having the structure

Including E and Z olefin isomers having the structure

4.1 methods for preparing Compounds of the invention

The compounds of the present invention can be prepared by conventional organic synthesis. For example, but not by way of limitation, compounds of the invention having formula I above can be prepared by the methods shown in schemes 1-4.

Scheme 1 shows how the compounds of formula I can be prepared by the Friedel-Crafts acylation method.

Scheme 1

The benzoyl chloride compound having formula 44 is coupled with a substituted or unsubstituted heterocyclic compound having formula 45 by Friedel-Crafts acylation (March, J.advanced organic chemistry-Reactions, Mechanisms and Structure, 4 th edition, John Wiley and Sons, New York, 1992, p. 539-542) to give an intermediate having formula 46. The compound of formula 46 is then reacted with a phosphine of formula 47 or a phosphonate of formula 48 in the presence of a base (e.g. lithium hexamethyldisilazide or potassium hexamethyldisilazide) by a Wittig reaction (March, J.advanced organic chemistry-Reactions, Mechanism and Structure, 4 th edition, John Wiley and Sons, New York, 1992, p. 956-963) to give the corresponding compound I.

In one embodiment, the base used in the Wittig reaction is LHMDS.

In another embodiment, the base used in the Wittig reaction is KHMDS.

General procedure A-Friedel-Crafts acylation

Aluminum trichloride (about 1 equivalent) is added to a 0.5 to 1.0M solution of a substituted or unsubstituted heterocyclic compound having formula 45 (about 1 equivalent) in methylene chloride at a temperature of 0 ℃. Benzoyl chloride having formula 44 (about 1 equivalent) was added to the resulting mixture and the reaction was allowed to warm to room temperature. The reaction mixture was then diluted with dichloromethane, washed with water (3 ×), dried over magnesium sulfate, filtered and concentrated in vacuo to give a crude residue. The crude residue was purified by flash column chromatography (hexane/ethyl acetate) to afford the compound having formula 46.

General procedure B-Wittig reaction

KHMDS (about 2 equivalents) is added to a 0.5 to 1.0M solution of the phosphine alkane having formula 47 or phosphonate having formula 48 (about 2 equivalents) in THF at a temperature of 0 deg.C. The resulting mixture was allowed to warm to room temperature and then stirred for about 15 minutes to about 1 hour, after which about 1.0M THF solution of the compound of formula 46 (about 1 equivalent) was added and the resulting mixture was heated to reflux for about 4 hours to about 36 hours while monitoring by TLC. The reaction was cooled to room temperature and then concentrated in vacuo to give a crude residue which was purified by flash column chromatography (hexane/ethyl acetate) to afford the compound of formula I.

Scheme 2 shows how the compounds of formula I are prepared by Grignard reaction.

Scheme 2

The bromobenzene or chlorobenzene having formula 43 is reacted with magnesium to prepare the corresponding Grignard reagent which is subsequently reacted with benzaldehyde having formula 44 to give the hydroxy compound having formula 45. The subsequent treatment of the hydroxy compound 45 with an oxidizing agent provides an intermediate compound having formula 40 which is reacted with a suitable phosphine 47 or phosphonate ester 48 in a Wittig reaction as described in scheme 1 above to provide a compound having formula I.

Suitable oxidizing agents for converting a compound having formula 45 to a compound having formula 40 include, but are not limited to, pyridinium chlorochromate (PCC), Pyridinium Dichromate (PDC), Jones reagent, Dess-Martin periodinane, MnO₂And tetra-n-propyl perruthenate (TPAP).

In a preferred embodiment, the oxidizing agent is PCC.

General procedure C-Grignard reaction

About 1/4 volumes of about 0.5M THF solution of the bromobenzene compound of formula 43 (about 1.2 equivalents) was slowly added to about 0.5M solution of magnesium turnings (about 1.2 equivalents) in THF. The resulting mixture is heated to reflux for about 30 minutes, after which the heat source is removed and the remainder of the bromobenzene compound of formula 43 is added dropwise. The resulting mixture was heated to reflux for about 5 hours to about 24 hours, then cooled to room temperature and stirred at room temperature for about 18 hours. The resulting solution was then added to about 0.5M THF solution of the benzaldehyde compound of formula 44 (about 0 deg.C) at a rate controlled so that the reaction temperature did not exceed 15 deg.C during the addition. After the addition was complete, the resulting reaction was stirred at room temperature for about 12 hours to about 24 hours, then cooled to about 0 ℃ and quenched with saturated aqueous ammonium chloride. The resulting mixture was extracted with ethyl acetate (3 ×), and the combined organic extracts were washed with water (3 ×) and brine, dried over magnesium sulfate and concentrated in vacuo to give a crude residue which was purified by flash column chromatography (hexane/ethyl acetate eluent) to afford the hydroxy compound having formula 45.

General procedure D-Oxidation of hydroxy Compounds having formula 45

Pyridinium chlorochromate (about 1.5 equivalents) and diatomaceous earth (about 100mg per 1mmol of the hydroxy compound having formula 45) are added to a solution of about 0.5M of the hydroxy compound having formula 45 (about 1 equivalent) in dichloromethane, and the resulting mixture is stirred for about 6 hours to about 24 hours. The reaction mixture was filtered, the resulting filter cake was washed with dichloromethane, the filtrate and washings were combined and concentrated in vacuo to give a crude residue which was purified by flash column chromatography to give the benzophenone compound of formula 40 which was converted to the compound of formula I by general method B described above.

Scheme 3 shows the synthesis of compounds having formula I by palladium catalyzed coupling of styrene and bromobenzene.

Scheme 3

General procedure Palladium catalyzed coupling of E-styrene and bromobenzene

About 0.5M Pd (OAc)₄(about 0.03 equivalents) of DMF solution was added to a suspension of DMF containing the styrene compound of formula 52 (about 1 equivalent), the bromobenzene compound of formula 53 (about 1.5 equivalents), sodium acetate (about 1.7 equivalents) and tetra-n-butylammonium bromide (about 1.1 equivalents). The resulting mixture was heated to 60 ℃ and stirred at this temperature for about 6 to about 18 hours, then cooled to room temperature and poured into a mixture of water ethyl acetate (3: 1). The organic phase was collected and the aqueous phase was washed with ethyl acetate (3 ×). The combined organic phases are washed successively with water and brine, then dried over magnesium sulfate, filtered and concentrated in vacuo to give a crude residue which is purified by flash column chromatography to give the compound of formula I.

Scheme 4 shows a method for preparing compounds having formula I using a phenyllithium intermediate.

Scheme 4

The reaction of a brominated compound having formula 49 with n-butyllithium affords the corresponding intermediate organolithium reagent which is then reacted with a benzaldehyde having formula 50 to afford the hydroxy compound having formula 51. The subsequent treatment of the hydroxy compound 51 with an oxidizing agent provides an intermediate compound having formula 46 which is reacted with a suitable phosphine or phosphonate 47 or 48 in a Wittig reaction as described in scheme 1 above to provide a compound having formula I.

Suitable oxidizing agents for converting a compound having formula 51 to a compound having formula 46 include, but are not limited to, pyridinium chlorochromate (PCC), Pyridinium Dichromate (PDC), Jones reagent, Dess-Martin periodinane, MnO₂And tetra-n-propyl perruthenate (TPAP).

In a preferred embodiment, the oxidizing agent is PCC.

One skilled in the art of organic chemistry knows how to prepare the compounds of the present invention using the methods described in schemes 1-4 and by simple chemical transformation of the products obtained by the methods of schemes 1-4.

Once synthesized, the compounds of the invention can be isolated from chemical precursors or other chemicals by standard purification techniques, such as chromatography (e.g., flash column chromatography and HPLC), asymmetric synthesis, recrystallization, and differential solubility.

4.2 Agents in combination with the Compounds of the invention

The invention provides methods of preventing, managing, treating, or ameliorating a disorder (e.g., a proliferative disorder, a disorder accompanied or characterized by aberrant angiogenesis, a disorder prevented, managed, or treated by inhibiting or reducing PDE4 expression and/or activity, or inhibiting or reducing tubulin polymerization and/or stability, or an inflammatory disorder), comprising administering to a subject in need thereof one or more compounds of the invention and one or more treatments (e.g., one or more prophylactic or therapeutic agents) other than a compound of the invention.

The invention also provides compositions comprising one or more compounds of the invention and one or more prophylactic or therapeutic agents other than a compound of the invention, and methods of using the compositions in the prevention, control, treatment, or amelioration of proliferative or inflammatory disorders. Prophylactic or therapeutic agents include, but are not limited to, small molecules, synthetic drugs, peptides, polypeptides, proteins, nucleic acids (e.g., DNA and RNA nucleotides including, but not limited to, antisense nucleotide sequences, RNAi, triplex and nucleotide sequences encoding biologically active proteins, polypeptides or peptides), antibodies, synthetic or natural inorganic molecules, mimetic agents (mimetopes), and synthetic or natural organic molecules.

Any agent known to be useful or already in use or currently being used to prevent, control, treat or ameliorate a disorder (e.g., a proliferative disorder, a disorder accompanied or characterized by aberrant angiogenesis, a proliferative disorder, an inflammatory disorder, a disorder prevented, controlled, treated or ameliorated by inhibiting PDE4 or reducing or inhibiting tubulin polymerization or stability) or one or more symptoms of a disorder can be used in combination with a compound of the invention described herein. For information on prophylactic or therapeutic agents that have been or are currently being used to prevent, treat, control or ameliorate a proliferative or inflammatory disorder or one or more symptoms thereof, see, e.g., Goodman and Gilman's by Gilman et al: the pharmacological basis of Therapeutics, 10 th edition, McGraw-Hill, New York, 2001, The Merck Manual of Diagnosis and Therapy (Merck Diagnosis and treatment Manual), BerkoW, M.D., et al, 17 th edition, Merck Sharp&Dohme Research Laboratories, Rahway, NJ, 1999, center Textbook of Medicine, 20 th edition, Bennett and Plum eds, W.B. Saunders, Philadelphia, 1996. Examples of such agents include, but are not limited to, anti-inflammatory agents Formulations (e.g., corticosteroids (e.g., prednisone and hydrocortisone), glucocorticoids, steroids, non-steroidal anti-inflammatory drugs (e.g., aspirin, ibuprofen, diclofenac, and COX-2 inhibitors), beta-agonists, anticholinergics, and methylxanthines), immunomodulators, aureolysine, sulfasalazine, penicillamine, antiangiogenic agents (e.g., angiostatin, TNF-alpha antagonists (e.g., anti-TNF-alpha antibodies), and endostatin), antifibrotic agents, antiemetics (e.g., metoclopramide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethenamide, ondansetron, granisetron, hydroxyzine, acetylleucine (acetoleucine) monoethanolamine, aripride, azasetron, benquinamide, bietanautine, bripride, chlorphenbutazine, clopride, clepride, clopride, and other anti-inflammatory drugs (e), such as anti-TNF-alpha antibodies, and endostatin), Benoxazine, dimenhydrinate, diphenidol, dolasetron, chlorphenazine, mesalamine, metopimazine, cannabilong, azapropazine, serphazine, scopolamine, sulpiride, tetrahydrocannabinol, thiethylperazine, thiopropazine and tropisetron), opioids (e.g., morphine, diacetylmorphine, hydromorphone, hydrocodone, oxymorphone, oxycodone, metoprolone, apomorphine, normorphine, tolorphine, buprenorphine, pethidine, loperamide, anileridine, isoxepinine, piminodine, beta-butopropionine, diphenoxylate, fentanyl (fentianil), sufentanil, alfentanil, remifentanil, levomorphine, dextromethorphan, pentazocine, cyclazocine, methadone, isoproxyprone and propoxyphene), colony stimulating factors (e.g., hematopoetic factor), clonorcinol, methadone, and prothromazine, PEGylated filgrastim (pegfilgrastim), sargrastim, moraxel and alfa-epoetin), antiemetics (e.g., metoclopramide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethoxy-benzamide, ondansetron, granisetron, hydroxyzine, acetylleucine (acetoleucine), monoethanolamine, aripride, azasetron, phenquine, theophylline, brompride, clofibrate, clobopride, phenmetrazine, dimenhydrinate, difenidol, dolasetron, chlorobenzazine, mesalamine, metopimazine, cannabirone, azapirone, serpimavamine, and alfa-ethyl-p-ethyl Oxazines, scopolamine, sulpiride, tetrahydrocannabinol, thiethylperazine, thioproperazine and tropisetron), dapsone, osteoprotegerin (e.g. methoxsalen and trimethadrine), antihistamines, antimalarial agents (e.g. hydroxychloroquine), antivirals, antibiotics (e.g. dactinomycin (previously known as actinomycin), bleomycin, erythromycin, penicillin, mithramycin and Apramycin (AMC)), vascular targeting agents (e.g. microtubule destabilizing drugs, combretastatin a-4 disodium phosphate, ZD6126, AVE8062, Oxi 4503, TZT 1027 and DMXAA), IMiD^®And SelCID^®(Celgene Corporation, New Jersey) (e.g., Revimid, Actimid and drugs disclosed in U.S. Pat. Nos. 6,075,041, 5,877,200, 5,698,579, 5,703,098, 6,429,221, 5,736,570, 5,658,940, 5,728,845, 5,728,844, 6,262,101, 6,020,358, 5,929,117, 6,326,388, 6,281,230, 5,635,517, 5,798,368, 6,395,754, 5,955,476, 6,403,613, 6,380,239 and 6,458,810, which are incorporated herein by reference).

4.2.1 immunomodulators

Any immunomodulator known to those skilled in the art may be used in the methods and compositions of the present invention.

Examples of immunomodulators include, but are not limited to, protein preparations, such as cytokines, peptidomimetic preparations, and antibodies (e.g., human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, Fv fragments, ScFv fragments, Fab fragments, or F (ab)₂Fragments or epitope-binding fragments), nucleic acid molecules (e.g., antisense nucleic acid molecules, triplexes, and nucleic acid molecules encoding immunomodulatory gene products), small molecules, organic compounds, and inorganic compounds. Specifically, immunomodulators include, but are not limited to, methotrexate (methothrexate), leflunomide, cyclophosphamide, cytoxan, Immuran, cyclosporin A, minocycline, azathioprine, antibiotics (e.g., FK506 (Tacrolimus)), Methylprednisolone (MP), corticosteroids, steroids (sterios), mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunomide), T cell receptor modulators, and cytokine receptor modulators.

Examples of cytokine receptor modulators include, but are not limited to, soluble cytokine receptors (e.g., the extracellular domain of a TNF- α receptor or fragments thereof, the extracellular domain of an IL-1 β receptor or fragments thereof, and the extracellular domain of an IL-6 receptor or fragments thereof), cytokines or fragments thereof (e.g., Interleukins (IL) -2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-23, TNF- α, TNF- β, Interferons (IFN) - α, IFN- β, IFN- γ, and GM-CSF), anti-cytokine receptor antibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (protein Designlabs)), anti-IL-4 receptor antibody, anti-IL-6 receptor antibody, anti-IL-10 receptor antibody, anti-IL-12 receptor antibody, anti-IL-15 receptor antibody and anti-IL-23 receptor antibody), anti-cytokine antibody (such as anti-IFN alpha antibody, anti-IFN beta antibody, anti-IFN gamma antibody, anti-TNF-alpha antibody, anti-IL-1 beta antibody, anti-IL-2 antibody, anti-IL-4 antibody, anti-IL-6 antibody, anti-IL-8 antibody (such as ABX-IL-8(Abgenix)), anti-IL-9 antibody, anti-IL-10 antibody, anti-IL-12 antibody and anti-IL-23 antibody). In particular embodiments, the cytokine receptor modulator is IL-4, IL-10, or a fragment thereof. In another embodiment, the cytokine receptor modulator is an anti-IL-1 β antibody, an anti-IL-6 antibody, an anti-IL-12 receptor antibody, or an anti-TNF- α antibody. In another embodiment, the cytokine receptor modulator is the extracellular domain of a TNF- α receptor, or a fragment thereof. In certain embodiments, the cytokine receptor modulator is not a TNF- α antagonist.

4.2.2 anti-angiogenic Agents

Any anti-angiogenic agent known to those skilled in the art may be used in the compositions and methods of the present invention. Non-limiting examples of anti-angiogenic agents include proteins, polypeptides, peptides, fusion proteins, antibodies (e.g., human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, Fv fragments, ScFv fragments, Fab fragments, F (ab))₂Fragments and antigen binding fragments thereof) such as immunospecific binding to TNF-Antibodies to alpha, nucleic acid molecules (e.g., antisense molecules or triple helices), organic molecules, inorganic molecules, and small molecules. Specifically, examples of anti-angiogenic agents include, but are not limited to, endothelial somatostatin, angiostatin, apomigren, anti-angiogenic antithrombin III, the 29kDa N-terminal and 40kDa C-terminal proteolytic fragments of fibronectin, uPA receptor antagonists, the 16kDa proteolytic fragment of prolactin, the 7.8kDa proteolytic fragment of platelet factor-4, the anti-angiogenic 24 amino acid fragment of platelet factor-4, the anti-angiogenic factor labeled 13.40, the anti-angiogenic 22 amino acid peptide fragment of thrombin sensitizing protein (thrombospondin) I, the anti-angiogenic 20 amino acid peptide fragment of SPARC, RGD and NGR-containing peptides, laminin, fibronectin, the anti-angiogenic peptides of procollagen and EGF, the anti-angiogenic peptides of anti-integrin alpha _vβ₃Antibodies, acidic fibroblast growth factor (aFGF) antagonists, basic fibroblast growth factor (bFGF) antagonists, Vascular Endothelial Growth Factor (VEGF) antagonists (e.g., anti-VEGF antibodies), and VEGF receptor (VEGFR) antagonists (e.g., anti-VEGFR antibodies).

Integrin alpha_vβ₃Examples of antagonists include, but are not limited to, protein preparations such as non-catalytic metalloprotease fragments, RGD peptides, peptidomimetics, fusion proteins, disintegrins or derivatives or analogs thereof, and immunospecific binding to integrin α_vβ₃The antibody of (a), a nucleic acid molecule, an organic molecule and an inorganic molecule. Immunospecific binding of integrin alpha_vβ₃Non-limiting examples of antibodies of (a) include 11D2 (Searle). Small molecule peptideometric integrin alpha_vβ₃Non-limiting examples of antagonists include S836(Searle) and S448 (Searle). Examples of disintegrins include, but are not limited to, Accutin. The present invention also includes any integrin alpha disclosed in the following U.S. patents and international application publications_vβ₃Use of antagonists in the compositions and methods of the invention: U.S. Pat. Nos. 5,652,109, 5,652,110, 5,578,704, 5,149,780, 5,196,511, 5,204,445, 5,262,520, 5,306,620, 5,478,725, 5,498,694, 5,523,209, 5,578,704, 5,589,570, 5,652,109, 5,652,110, 5,693,612, 5,705,481, 5,753,230, 5,767,071, 5,770,565, 5,780,426, 5,817,457, 5,830,678, 5,849,692, 5,955,572, 5,985,278, 6,048,861, 6,090,944, 6,096,707, 6,130,231, 6,153,628, 6,160,099 and 6,171,58, as well as international application publications WO 95/22543, WO 98/33919, WO 00/78815, WO 00/31248, WO 98/46264, WO 98/40488 and WO 02/070007, each of which is incorporated herein by reference.

In a particular embodiment of the invention, the anti-angiogenic agent is endostatin. Natural endostatin consists of C-terminal-180 amino acids of collagen XVIII (cDNA encoding two splice forms of collagen XVIII, with GenBank accession numbers AF18081 and AF 18082). In another embodiment of the invention, the anti-angiogenic agent is a plasminogen fragment (coding sequence for plasminogen is found in GenBank accession nos. NM — 000301 and a 33096). Angiostatin peptides naturally include the four kringle domains (kringle 1 through kringle4) of plasminogen. Recombinant kringles 1, 2 and 3 have been shown to have the anti-angiogenic properties of native peptides, whereas kringle4 does not have this activity (Cao et al, 1996, J.biol.chem.271: 29461-29467). Thus, the cytosomatostatin peptide includes at least one, preferably more than one kringle domain selected from kringle 1, kringle 2 and kringle 3. In a specific embodiment, the anti-angiogenic peptide is a 40kDa isoform of a human angiostatin molecule, a 42kDa isoform of a human angiostatin molecule, a 45kDa isoform of a human angiostatin molecule, or a combination thereof. In another embodiment, the anti-angiogenic agent is the kringle 5 domain of plasminogen, which is a more potent inhibitor of angiogenesis than angiostatin (a kringle 1-4 domain containing angiostatin). In another embodiment of the invention, the anti-angiogenic agent is antithrombin III. Antithrombin III (hereinafter antithrombin) comprises a heparin-binding domain that binds proteins to the vessel wall and an active site loop that interacts with thrombin. When antithrombin is bound to heparin, the protein elicits a conformational change that allows the active loop to interact with thrombin, resulting in proteolytic cleavage of the loop by thrombin. Proteolytic cleavage events result in another change in the conformation of antithrombin, namely (i) a change in the same interaction interface between thrombin and antithrombin, and (ii) release of the complex from heparin (Carrell, 1999, Science 285: 1861-1862, and references therein). O' Reilly et al (1999, Science 285: 1926-1928) have found that the dissociated antithrombin has strong anti-angiogenic activity. Thus, in one embodiment, the anti-angiogenic agent is an anti-angiogenic form of antithrombin. In another embodiment of the invention, the anti-angiogenic agent is a 40kDa and/or 29kDa proteolytic fragment of fibronectin.

In another embodiment of the invention, the anti-angiogenic agent is a urokinase plasminogen activator (uPA) receptor antagonist. In one embodiment, the antagonist is a dominant negative mutation of uPA (see, e.g., (Crowley et al, 1993, Proc. Natl. Acad. Sci. USA 90: 5021-, An anti-angiogenic factor, anti-angiogenic 22 amino acid peptide fragment of thrombin sensitin (thrombin) I, anti-angiogenic 20 amino acid peptide fragment of SPARC, anti-angiogenic small peptide of laminin fibronectin, procollagen or EGF, or integrin alpha labeled 13.40 _vβ₃Or a small peptide antagonist of the VEGF receptor. In another embodiment, the small peptide comprises an RGD or NGR motif. In certain embodiments, the anti-angiogenic agent is a TNF- α antagonist. In other embodiments, the anti-angiogenic agent is notAre TNF-alpha antagonists.

According to the methods of the invention, a nucleic acid molecule encoding a protein, polypeptide or peptide having anti-angiogenic activity, or a protein, polypeptide or peptide having anti-angiogenic activity, can be administered to an individual suffering from a disorder (e.g., a disorder characterized by or accompanied by aberrant angiogenesis, a proliferative disorder, an inflammatory disorder, or a disorder prevented, controlled, treated or ameliorated by inhibition of PDE4, or by reduction or inhibition of tubulin polymerization or stability). Furthermore, according to the methods of the present invention, a nucleic acid molecule encoding a derivative, analog, fragment or variant of a protein, polypeptide or peptide having anti-angiogenic activity, or a derivative, analog, fragment or variant of a protein, polypeptide or peptide having anti-angiogenic activity, can be administered to an individual having a disorder (e.g., a disorder characterized by or accompanied by aberrant angiogenesis, a proliferative disorder, an inflammatory disorder, or a disorder prevented, controlled, treated or ameliorated by inhibition of PDE4 or by reduction or inhibition of tubulin polymerization or stability). Preferably, such derivatives, analogs, variants and fragments retain the anti-angiogenic activity of the full-length wild-type protein, polypeptide or peptide.

Proteins, polypeptides or peptides useful as anti-angiogenic agents can be prepared by any of the techniques well known in the art or described herein. Proteins, polypeptides or peptides having anti-angiogenic activity can be engineered by techniques well known in the art or described herein to increase the in vivo half-life of such proteins, polypeptides or peptides. Commercially available anti-angiogenic agents are preferably used in the compositions and methods of the present invention. The anti-angiogenic activity of the formulation can be determined in vitro and/or in vivo by any technique known to those skilled in the art or described herein.

Anti-angiogenic agents and their dosages, routes of administration, and recommended use are known in the art and have been described in literature, for example, in the physicians' Desk Reference (57 th edition, 2003).

4.2.3 TNF-alpha antagonists

Any TNF- α antagonist known to those skilled in the art may be used in the compositions and methods of the present invention. Non-limiting examples of TNF- α antagonists include proteins, polypeptides, peptides, fusion proteins, antibodies (e.g., human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, Fv fragments, ScFv fragments, Fab fragments, F (ab)) ₂Fragments and antigen-binding fragments thereof) such as antibodies that immunospecifically bind TNF- α, nucleic acid molecules (e.g., antisense molecules or triplexes), organic molecules, inorganic molecules, and small molecules. In various embodiments, the TNF- α antagonist reduces the function, activity, and/or expression of TNF- α by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to a control, e.g., Phosphate Buffered Saline (PBS).

Examples of antibodies that immunospecifically bind TNF- α include, but are not limited to, infliximab (REMICADE ®; Centacor), D2E7(Abbott Laboratories/Knollpharmaceutical Co., Mt. Olive, N.J.), CDP571 (also known as HUMICAE)^TM) And CDP-870 (both from Celltech/Pharmacia, Slough, U.K.) and TN₃19.12(Williams et al, 1994, Proc. Natl. Acad. Sci. USA 91: 2762-. The invention also includes the use of antibodies that immunospecifically bind TNF- α, which antibodies are disclosed in the following U.S. patents: U.S. patent nos. 5,136,021, 5,147,638, 5,223,395, 5,231,024, 5,334,380, 5,360,716, 5,426,181, 5,436,154, 5,610,279, 5,644,034, 5,656,272, 5,658,746, 5,698,195, 5,736,138, 5,741,488, 5,808,029, 5,919,452, 5,958,412, 5,959,087, 5,968,741, 5,994,510, 6,036,978, 6,114,517, and 6,171,787. Each of which is incorporated herein by reference. Examples of soluble TNF-alpha receptors include, but are not limited to, sTNF-R1(Amgen), etanercept (ENBREL) ^TM；Immunex) And its rat homolog RENBERL^TMSoluble inhibitors of TNF- α derived from TNFR I, TNFR II (Kohno et al, 1990, Proc. Natl. Acad. Sci. USA 87: 8331-) -8335) and TNF- α Inh (Seckinger et al, 1990, Proc. Natl. Acad. Sci. USA 87: 5188-) -5192).

In one embodiment, the TNF- α antagonist used in the compositions and methods of the present invention is a soluble TNF- α receptor. In a particular embodiment, the TNF- α antagonist used in the compositions and methods of the invention is etanercept (ENBREL)^TM(ii) a Immunex) or a fragment, derivative or analogue thereof. In another embodiment, the TNF- α antagonists used in the compositions and methods of the present invention are antibodies that immunospecifically bind TNF- α. In a particular embodiment, the TNF- α antagonist used in the compositions and methods of the present invention is infliximab (REMICADE ®; Centacor), a derivative, analog, or antigen-binding fragment thereof.

Other TNF- α antagonists encompassed by the invention include, but are not limited to, IL-10 (which is known to block TNF- α production by interferon γ -activated macrophages (Oswald et al 1992, Proc. Natl. Acad. Sci. USA 89: 8676-, tenadap (enablex), melanin (Large Scale Biological), and anti-p 38MAPK formulation (Uriach).

TNF- α antagonists and their dosages, routes of administration, and recommended use are known in the art and have been described in, for example, the Physician's Desk Reference (57 th edition, 2003).

4.2.4 anti-inflammatory Agents

Anti-inflammatory agents have been successful in the treatment of proliferative or inflammatory disorders and are now common and standard treatments for such disorders, as well as other disorders. Any anti-inflammatory therapy (e.g., anti-inflammatory agents) well known to those skilled in the art may be used in the compositions and methods of the present invention. Non-limiting examples of anti-inflammatory agents include various non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs, beta-agonists, anticholinergic (anticholinergic) agents, antihistamines (e.g., ethanolamine, ethylenediamine, piperazine, and phenothiazine), and methylxanthines. Examples of NSAIDs include, but are not limited to, aspirin, ibuprofen, salicylate, acetominophen, Celecoxib (CELEBREX)^TM) Diclofenac (VOLTAREN)^TM) Etodolac (LODINE)^TM) Fenoprofen (NALFON)^TM) Anti-inflammatory pain (INDOCIN)^TM) Ketorolac (ketoralac) (TORADOL)^TM) Oxaprozin (DAYPRO)^TM) Nabumetone (nalbupone) (RELAFEN)^TM) Sulindac (CLINORIL)^TM) Tolmetin (TOLECTIN)^TM)、rofecoxib(VIOXX^TM) Naproxen (ALEVE) ^TM，NAPROSYN^TM) Ketoprofen (ACTRON)^TM) And Nabumetone (RELAFEN)^TM). Such NSAIDs act by inhibiting cyclooxygenase enzymes (e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatory drugs include, but are not limited to, glucocorticoids, dexamethasone (DECADRON)^TM) Cortisone, hydrocortisone, prednisone (Deltasine)^TM) Prednisolone, triamcinolone, salicylazosulfapyridine (azulfidine), and eicosanoids (e.g., prostaglandins, thromboxanes, and leukotrienes).

Anti-inflammatory agents and their dosages, routes of administration, and recommended use are known in the art and have been described in, for example, the Physician's Desk Reference (57 th edition, 2003).

4.2.5 anticancer Agents

Any treatment (e.g., prophylactic or therapeutic) known to be useful, used, or currently being used to prevent, treat, control, or ameliorate one or more symptoms associated with a proliferative disorder (e.g., cancer) can be used in the compositions and methods of the invention. Prophylactic or therapeutic agents include, but are not limited to, peptides, polypeptides, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules. Non-limiting examples of cancer therapy include chemotherapy, radiation therapy, hormonal therapy, and/or biological/immunotherapy.

In certain embodiments, the anti-cancer agent is an immunomodulatory agent, such as a chemotherapeutic agent. In other embodiments, the anti-cancer agent is not an immunomodulatory agent. In a particular embodiment, the anti-cancer agent is an anti-angiogenic agent. In other embodiments, the anti-cancer agent is not an anti-angiogenic agent.

Examples of anti-cancer agents include, but are not limited to: acivicin, aclarubicin, alcaroxazole hydrochloride, alclomezine, aldehydamine, aldesleukin, altretamine, ambomycin, amicridine, anastrozole, amtriptolide, asparaginase, clindamycin, azacitidine, azatepa, azamycin, batide, benzofenamipide, bicalutamide, bisnadide dimesylate, bisphosphonates (e.g., pamidronate (Aredria), sodiumclonate (Bonefos), zoledronic acid (Zopita), alendronate (Fosamax), etidronate, ibandonate, cimadronate, risedronate and tiludronate), bizedroxin, bleomycin, alexan, testosterone, piroctone, beclomezine, mechlorethamine, azamethicillin, azalide, azamethicillin, mechlorethamine, mec, Casubicin hydrochloride, cabenzepine, cidofol, chlorambucil, siromycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin hydrochloride, decitabine, dexomaplatin, dizonganine, dizagutanine mesylate, diazaquinone, docetaxel, doxorubicin hydrochloride, droloxifene citrate, drostandrostarone propionate, diazomycin, edatrexate, eflornithine hydrochloride (eflornithine), elsamitrucin, enrofloxacin, epipirimidine hydrochloride, epirubicin hydrochloride, erbuzole, esorubicin hydrochloride, estramustine sodium phosphate, etanidazole, etoposide phosphate, etoprine phosphate, favudine hydrochloride, favudine, fazab, fludarabine A, fludarabine phosphate, fludarabine hydrochloride, fludarabine hydrochloride, fludarabine, Phosphaquizalone, forstrocin sodium, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, imofovir, interleukin-2 (including recombinant interleukin 2 or rIL2), interferon alpha-2 a, interferon alpha-2 b, interferon alpha-nl, interferon alpha-n 3, interferon beta-Ia, interferon beta-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liazole hydrochloride, lometrexol sodium, lomustine, loxoanthraquinone hydrochloride, maxol, maytansine, mechlorethamine hydrochloride, anti-CD 2 antibodies, megestrol acetate, melengestrol acetate, melphalan, melnolle, mercaptopurine, methotrexate sodium, metoline, metopipamide, mitochodrine, mitochodrin, mitochocin, mitochomycin, medroxobin, medetomycin, Mitomycin, mitospirane, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogomycin, omaplatin, oxsulam, paclitaxel, pemetrexed, pelithromycin, neostigmine bromide, pelothromycin sulfate, perphosphamide, pipobroman, piposulfan, piroanthraquinone hydrochloride, plicamycin, plurameltem, porfimer sodium, podofycin, prednimustine, procarbazine hydrochloride, puromycin hydrochloride, pyrazolofuranin, lyboadenosine, roguinimine, safranol, saffingo hydrochloride, semustine, octreozine, spartate sodium, sparpamycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulfochlorambucil, talamycin, tecolandium, gazoxogum, gatifloxacin hydrochloride, telocine, teniposide, tipepidopurine, testotene, epididymidone, guanylne, guanidium, tiacumicine, tiadinin, testosterone, guanicine, tiamulin, guanicine, tia, Thiotepa, thiazolfurin, tirapazamine, toremifene citrate, tritulone acetate, triciribine phosphate, trimetrexate, tritrexate-glucuronate, triptorelin, tobrozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinrosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, sethoxydim and zorubicin hydrochloride.

Other anti-cancer agents include, but are not limited to: 20-epi-1, 25-dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, acyclopentanol (adecanol), aldeoxin, aldesleukin, ALL-TK antagonist, hexamethamine, ammostatin, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitor, antagonist D, antagonist G, antarelix, anti-dorsal morphogenetic protein-1, anti-androgen (prostate cancer), anti-estrogen, anti-canotone (antineoplaston), antisense oligonucleotide, glycine aphidicolin, apoptosis gene regulator, apoptosis regulator, depurination regulator, apurinic nucleic acid, ara-CDP-DL-PTBA, arginine deaminase, asulacrine, atamestane, amoxastine, axiastatin 1, axiastatin 2, axiastatin 3, azastan 3, azastan, azatoxin, diazotyrosine, baccatin III derivatives, balanol, batimastat, BCR/ABL antagonists, benzodichlorine, benzoylstaphin, beta-lactam derivatives, beta-alethine, betaclamycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisaziridinyl spermine, bisnafide, bistetralene A, bizelesin, brefellate, bripirimid, bricropin, buthionine sulfoximine (buthionine sulfoximine), calcipotriol, carboplatin C, camptothecin derivatives, canarypox IL-2, capecitabine, carboxamide-amino-triazole, carboxyamidotriazole, CaRest M3, CARN 700, cartilage derived inhibitors, calcilezelesin kinase Inhibitors (ICOS), caspoetin, cecromoline B, cetroridine, quinoxaline, chlotrimazine, clotrimine, chlozine, chloprene, cystamide, prostacyclin analogs, betrinine, betrinol, bemisin 700, chondrosine, cystatin, clotrimine, cystatin, and other analogs, Clotrimazole, colimycin A, colimycin B, combretastatin A4, combretastatin analogs, coagenin, crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curainA, cyclopentaquinone, cycloplatam, cyclopemycin, cyclabine, cytarabine, cytolysfamate, cytostatin, dacliximab, decitabine, dehydromembranocembrin B, delorelin, dexamethasone, dexfosfamide, dexrazolamide, dexverapamil, dexrazoxane, hymexazol B, didox, diethylspermidine, dihydro-5-azacytidine, 9-dihydrotaxol, dioxamycin, diphenylomostin, docetaxel, docosanol, dol, fluazuron, fluxolone, valacyclin, elenide, isolatrine, epirubicin B, epirubicin, etidronabine, etidronide, epirubicin, etidronedarone, doxorubicin, doxycycline, domorphin, nalin B, valacyclobetamethamphetacin, valacyclofibrate, etidine, etidronabine, etidine, etidronabine, etidine, etidronide, etidine, etidronabine, etidine, etimiine, doxycycline, Etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgramine, finasteride, flavopiridol, fludrostine, flusterone, fludarabine, fluurodoranuronicin hydrochloride, folamectin, formestane, forskocin, fotemustine, gadolinium texaphyrin, gallium nitrate, galotabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, HMG CoA reductase inhibitors (e.g., atorvastatin, cerivastatin, fluvastatin, lescol, lupittor, lovastatin, rosuvastatin and simvastatin), hepsulfamam, heregulin, hexamethylenebisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene, soramanib, amantane, ivastatin, idarubicin, fluvastatin, fludarabicin, an agonist, an interferon-4 interferon, an interferon-4-interferon, 4-related to 4-subunit antagonists, such as, a, iroplat, issorafen, isobengazole, isoomohalidondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, leguminomycin, lentinan sulfate, leptin, letrozole, leukemia inhibitory factor, leukocyte interferon alpha, leuprolide + estrogen + progesterone, leuprolide, levamisole, LFA-3TIP (Biogen, Cambridge, MA; U.S. Pat. No. 6,162,432), linazole, linear polyamine analogs, lipopeptide diglycol, lipophilic platinum compounds, lissorulin amide 7, lobaplatin, earthworm phospholipid, lometrexol, lonidamine, losoxantrone, loxoribine, luratetropin, lyxophyrine, lysinomycin, maytansine, mazinostatin, methoprimidol, mestamide, mestamarine, mestamide, mestamarine, mes, Miltefosine, milbemycin, mismatched double-stranded RNA, mitoguazone, dibromodulcitol, mitomycin analogs, mitonaphthylamine, mitotoxin fibroblast growth factor-saporin, mitoxantrone, moffarantine, moraxetin, monoclonal antibodies, human chorionic gonadotropin, azophospolipid A + Mycobacterium labrochelle, mopidanol, inhibitors of multiple drug resistant genes, multiple tumor inhibitor-based therapies, mustard anticancer agents, mycaperoxide B, mycobacterial cell wall extracts, myriaperone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagarethip, naloxone + pentazocine, napavirin, napterin, nartostatin, napraplatin, nemorubicin, nerubicin, neridronic acid, neutral endopeptidase, nilutamide, nisin, nitric oxide modulators, nitrotrinitrosine, guanixin, guanixine, octreotide, and other drugs, Onapristone, ondansetron, oracin, an oral cytokine inducer, ormaplatin, oxalirone, oxaliplatin, oxaauromycin, paclitaxel analogs, paclitaxel derivatives, palauamine, palmitoyldizoxin, pamidronic acid, panaxytriol, panomifene, paraabactin, paclobutin, pemphidase, peldesine, pentosan polysulfate, pentostatin, pendrazole, perfluorobromoalkane, perphosphamide, perillyl alcohol, phenazinomocin, phenyl acetate, a phosphatase inhibitor, a streptolysin preparation, pilocarpine hydrochloride, pirarubicin, pirtrocrine, placetin A, placetin B, a plasminogen activator inhibitor, a platinum complex, a platinum compound, a platinum-triamine complex, a porphin sodium, a Pofimicin, prednisone, propyldicridone, acridinone J, an 2, an inhibitor of an immune protein kinase, a protein C protein-based protein kinase inhibitor, a protein C protein kinase inhibitor, microalgal, protein tyrosine phosphatase inhibitor, purine nucleoside phosphorylase inhibitor, rhodopsin, pyrazoloraridine, pyridoxylated hepatoglobin polyoxin polyoxythienylene conjugate, raf antagonist, raltitrexed, ramustine, ras farnesyl protein transferase inhibitor, ras-GAP inhibitor, retipine methylated, etidronate rhenium Re 186, rhizoxin, ribozyme, RII retinamide, luvulcamide, rohitume, romopeptide, roquinmerac, rubiginone B1, ruboxyl, saragophyllin, saintopin, SarCNU, saropotol A, sargrastim, Sdi1 mimetic, semustine, senescence-derived inhibitor 1, oligonucleotide, signal transduction modulator, signal chain antigen binding protein, growth promoting protein, spigothin, tretinomycin, stiiamide, stromelysin inhibitors, sulfinosine, superactive vasoactive intestinal peptide antagonists, suradista, suramin, stanin, synthetic glycosaminoglycans, tamoxifen, 5-fluorouracil, leucovorin, tamoxifen methiodide, taurolidine, tazarotene, tecogalan sodium, tegafur, telluropyrylium, telomerase inhibitors, temoporfin, temozolomide, teniposide, tetrachlorecaoxide, tetratramamine, thalibrostatin, thiocoraline, thrombopoietin mimetic, thymolfasin, thymopoietin receptor agonist, thymotreonam, thyroid stimulating hormone, ethinyl ethylpuriin, tirazamine, dicyclopentylpeptide, tolsententin, toremifene, dry cell factor, translation inhibitors, tretinosyl, treonamide, tretinospora, tyrosine, treonamide, tretinospora, tretinomycin, trexone, tremulin, UBC inhibitor, ubenimex, urogenital sinus-derived growth inhibitor, urokinase receptor antagonist, vapreotide, variolin

B. Vector systems (erythrocyte gene therapy), thalidomide, veratrilol, veramine, verdins, verteporfin, vinorelbine, vinxaline, vorozole, zanoterone, zeniplatin, benzalkonium and zinostatin stimamer.

In a more specific embodiment, the invention also includes the administration of a compound of the invention in combination with one or more therapies (such as, but not limited to, the anti-cancer agents disclosed in table 2), preferably for the treatment of breast, ovarian, melanoma, prostate, colon, and lung cancers.

TABLE 2

Therapeutic formulations	Dosage/administration mode/dosage form
				Doxorubicin hydrochloride (doxorubicin RDF ® and doxorubicin PFS ®)	Vein	60-75mg/m on day 12	At intervals of 21 days
Epirubicin hydrochloride (Ellence)TM)	Vein	100-120mg/m on day 1 of each cycle2Or uniformly distributed and administered on days 1-8 of the cycle	3-4 week period
				Fluorouracil	Vein	The specification of the medicine is as follows: vials 5ml and 10ml (containing 250 and 500mg fluorouracil, respectively)
Docetaxel (Taxotere ®)	Vein	60-100mg/m within 1 hour2	Every 3 weeks for 1 time
				Palitaxi (Taxol ®)	Vein	175mg/m in 3 hours2	For a total of 4 courses, 1 time every 3 weeks (sequential administration with combination chemotherapy containing doxorubicin)
Tamoxifen citrate (Nolvadex ®)	Oral administration (tablet)	The dosage of 20-40mg more than 20mg should be administered as divided doses (morning and evening)	Daily life
				Calcium folinate for injection	Intravenous or intramuscular injection	The specification of the medicine is as follows: 350mg small bottle	Text unspecified dose PDR 3610
luprolide acetate(Lupron®)	Single dose subcutaneous injection	1mg (0.2mL or 20 units mark)	1 time per day
				Flutamide (Eulexin ®)	Oral administration (Capsule)	250mg (125 mg flutamide per capsule)	At 8-hour intervals 3 times a day (total daily dose 750mg)
Nilutamide (Nilandron ®)	Oral administration (tablet)	300mg or 150mg (each tablet containing 50 or 150mg nilutamide)	300mg daily for 30 days, followed by 150mg once daily
				Bicalutamide (Casodex ®)	Oral administration (tablet)	50mg (each tablet contains 50mg bicalutamide)	1 time per day
Progesterone	Injection of drugs	USP 50mg/ml sesame oil
				Ketoconazole (Nizoral ®)	Cream	The 2% cream is applied 1 or 2 times per day according to symptoms
Prednisone	Oral administration (tablet)	The initial dose may be from 5mg to 60 mg/day, depending on the particular disease being treated

		Disease entity
				Estramustine sodium phosphate (Emcyt ®)	Oral administration (Capsule)	14mg/kg body weight (i.e. one 140mg capsule per 10kg or 221b body weight)	Administering 3 or 4 divided doses per day
Etoposide or VP-16	Vein	5mL of a 20mg/mL solution (100mg)
				Dacarbazine (DTIC-Dome ®)	Vein	2-4.5mg/kg	1 time per day for 10 days. Can be repeated at intervals of every 4 weeks
Polifeprosan 20 carmustine implant (BCNU) (nitrosourea) (Gliadel ®)	Placing wafer in the excision Cavity	If the size and shape of the resection cavity allows, 8 wafers, each containing 7.7mg carmustine, were placed together61.6mg
				Cis-platinum	Injection of drugs	[ n/a in PDR 861]The specification of the medicine is as follows: 1mg/ml solution in 50ml and 100ml multidose vials
Mitomycin	Injection of drugs	Supplied in 5mg and 20mg vials (containing 5mg and 20mg mitomycin)
				Gemcitabine hydrochloride (Gemzar ®)	Vein	NSCLC-2 regimens have been studied but no optimal regimen has been determined for 4 week regimens-30 min intravenous administration of 1000mg/m23 week protocol-Gemzar 1250mg/m administered intravenously over 30 minutes2	4 week protocol-days 1, 8 and 15 of each 28 day cycle. Day 1 after Gemzar injection at 100mg/m2Cisplatin was administered intravenously for a 3-week schedule-days 1 and 8 of each 21-day cycle. Day 1 after Gemzar injection at 100mg/m2Intravenous cisplatin administration
Carboplatin (Paraplatin ®)	Vein	Single-preparation treatment: 360mg/m on day 12V. (infusion lasted 15 minutes or longer) other dose calculations: combination therapy with cyclophosphamide, recommended dose adjustment, prescribed dose, etc.	Every 4 weeks
				Ifosamide(Ifex®)	Vein	1.2 g/m/day2	Repeating every 3 weeks for 5 consecutive days or after recovery from hematologic toxicity
Topotecan hydrochloride	Vein	1.5mg/m per day2Intravenous injection 30	From day 1 of the 21 course of treatment

(Hycamtin®)

Minute (min)

The day is continuously for 5 days

In particular embodiments, radiation therapy (including the use of x-rays, gamma rays, and other radioactive sources to destroy cancer cells) is used in combination with the antibodies of the present invention. In a preferred embodiment, radiation therapy is delivered by external radiation beam or remote radiation therapy, wherein the radiation is from a remote source. In other preferred embodiments, radiation therapy is delivered as internal or brachytherapy, in which the radiation source is placed in proximity to cancerous cells or tumor tissue in vivo.

Cancer treatments and their dosages, routes of administration, and recommended use are known in the art and have been described in, for example, the Physician's Desk Reference (57 th edition, 2003).

4.2.6 antibiotic

Antibiotics well known to those skilled in the art may be used in the compositions and methods of the present invention. Non-limiting examples of antibiotics include penicillin, cephalosporin, imipenem, axtreonam, vancomycin, cycloserine, bacitracin, chloramphenicol, erythromycin, clindamycin, tetracycline, streptomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin, spectinomycin, trimethoprim, norfloxacin, rifampin, polymyxin, amphotericin B, nystatin, ketocanazole, isoniazid, metronidazole, and pentamidine.

Antibiotics and their dosages, routes of administration, and recommended uses are known in the art and have been described in, for example, the Physician's Desk Reference (57 th edition, 2003).

4.2.7 antiviral preparation

Any antiviral agent known to those skilled in the art may be used in the compositions and methods of the present invention. Non-limiting examples of antiviral agents include proteins, polypeptides, peptides, fusion protein antibodies, nucleic acid molecules, organic molecules, inorganic molecules, and small molecules that inhibit or reduce binding of a virus to its receptor, internalization of a virus into a cell, replication of a virus, or release of a virus from a cell. Specifically, antiviral agents include, but are not limited to, nucleoside analogs (e.g., zidovudine, acyclovir, gancyclovir, vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine, rimantadine, saquinavir, indinavir, ritonavir, interferon-alpha, and other interferons, and AZT.

Antiviral formulations and dosages, routes of administration, and recommended use thereof are known in the art and have been described in, for example, the Physician's Desk Reference (56 th edition, 2002).

4.2.8 vascular targeting formulations

Any vascular targeting agent known to those of skill in the art may be used in the compositions and methods of the present invention (see, e.g., Thorpe, p.e., clin.can.res.10: 415-427(2004), which is incorporated herein by reference).

Non-limiting examples of vascular targeting agents include small molecule vascular targeting agents (e.g., microtubule destabilizing drugs, combretastatin a-4 disodium phosphate, ZD6126, AVE8062, Oxi 4503, TZT 1027, and DMXAA) and ligand-based vascular targeting agents, including but not limited to fusion proteins (e.g., vascular endothelial growth factor linked to the phytotoxin gelonin), immunotoxins (e.g., monoclonal antibodies to endoglin conjugated to ricin a), cytokine-linked antibodies, and liposome-encapsulated drugs.

In one embodiment, the ligand-based vascular targeting agent includes any ligand that selectively binds to a component of a tumor vessel linked (e.g., by a chemical cross-linker or peptide bond) to an agent capable of occluding a tumor vessel. Examples of ligands that selectively bind to tumor vascular components include, but are not limited to, antibodies or peptides directed against markers that are selectively upregulated in tumor tissue endothelial cells as compared to normal tissue endothelial cells. Examples of markers that are selectively upregulated on endothelial cells in tumor tissue as compared to endothelial cells in normal tissue include, but are not limited to, inflammatory mediator (e.g., Interleukin (IL) -1) -induced cell adhesion molecules and molecules that accompany hypercoagulable changes that occur in the endothelium of tumor vessels. Examples of agents capable of occluding tumor vessels include, but are not limited to, coagulation-inducing proteins (e.g., tissue factor), toxins (e.g., diphtheria toxin, ricin, gelonin), cytotoxic agents (e.g., doxorubicin, neocarzinostatin), cytokines (e.g., interleukin-2, interleukin-12, tumor necrosis factor-alpha), apoptosis-inducing agents (e.g., RAF-1 gene, mitochondrial-membrane disruptive peptide), radioisotopes (e.g., iodine-131, actinium-225, bismuth-213), and liposome-encapsulated effectors (e.g., cytosine arabinoside derivatives).

Vascular targeting agents and their dosages, routes of administration and recommended use are known in the art and have been described in the literature, for example, in the physicians' Desk Reference (57 th edition, 2003).

4.3 use of the Compounds of the invention

The present invention relates to therapeutic methods for the prevention, treatment, control, or amelioration of a disease or disorder, or one or more symptoms thereof, by administering one or more compounds of the invention, or compositions comprising such compounds, to a subject, preferably a human subject. In one embodiment, the invention provides a method of preventing, treating, managing or ameliorating a disease or disorder or one or more symptoms thereof, the method comprising administering to an individual in need thereof a prophylactically or therapeutically effective amount of one or more compounds of the invention.

The invention also provides methods of preventing, treating, managing or ameliorating a disease or disorder or one or more symptoms thereof, comprising administering to a subject in need thereof one or more compounds of the invention and one or more therapies (e.g., one or more prophylactic or therapeutic agents) currently in use, known or used to prevent, treat or ameliorate one or more symptoms associated with the disease or disorder. The prophylactic or therapeutic formulations of the combination therapies of the invention may be administered sequentially or simultaneously. In a particular embodiment, the combination therapy of the invention comprises one or more compounds and at least one other therapy (e.g. another prophylactic or therapeutic agent) having the same mechanism of action as the compound. In another specific embodiment, the combination therapy of the invention comprises one or more compounds of the invention and at least one other therapy (e.g., another prophylactic or therapeutic agent) having a different mechanism of action than the compound. In certain embodiments, the combination therapies of the invention enhance the prophylactic or therapeutic effect of one or more compounds of the invention by co-acting with each compound to achieve an additive or synergistic effect. In certain embodiments, the combination therapies of the invention reduce the side effects associated with treatment (e.g., prophylactic or therapeutic agents).

The prophylactic or therapeutic formulations of the combination therapy can be administered to the individual (preferably a human individual) in the same pharmaceutical composition. In alternative embodiments, the prophylactic or therapeutic formulations of the combination therapy may be administered to the individual simultaneously in different pharmaceutical compositions. The prophylactic or therapeutic formulations may be administered to the individual by the same or different routes of administration.

In a particular embodiment, a pharmaceutical composition comprising one or more compounds of the invention is administered to an individual (preferably a human) to prevent, treat, control or ameliorate one or more symptoms associated with the disease or disorder. According to the present invention, the pharmaceutical compositions of the present invention may further comprise one or more prophylactic or therapeutic agents currently in use, used, or known to be useful for preventing, treating, or ameliorating one or more symptoms associated with a disease or disorder.

Diseases or conditions that may be prevented, treated, controlled or ameliorated by the administration of an effective amount of one or more compounds of the invention include, but are not limited to, conditions characterized by or accompanied by abnormal angiogenesis, central nervous system conditions, proliferative conditions, inflammatory conditions, autoimmune conditions, conditions that are prevented, controlled, treated or ameliorated by vascular inhibition (e.g., blocking angiogenesis by vascular inhibition), and conditions that are prevented, controlled, treated or ameliorated by inhibiting and/or reducing the expression and/or activity of PDE4 or by inhibiting or reducing the polymerization or stability of tubulin. Examples of disorders characterized by or accompanied by angiogenesis include, but are not limited to, proliferative disorders (e.g., cancer). Examples of conditions that are prevented, controlled, treated or ameliorated by inhibiting or reducing the expression and/or activity of PDE4 include, but are not limited to, inflammatory conditions such as asthma, inflammation, chronic or acute lung obstructive disease, chronic or acute pulmonary inflammatory disease, inflammatory bowel disease, crohn's disease, behcet's disease, HSP, colitis and reperfusion-induced inflammation. Examples of conditions that are prevented, controlled, treated or ameliorated by inhibiting or reducing the polymerization or stability of tubulin include, but are not limited to, proliferative conditions (e.g., cancer) and non-cancerous conditions (e.g., psoriasis and fibrosis).

In a particular embodiment, the invention provides a method of preventing, managing, treating or ameliorating a disease or condition, including a condition that is prevented, managed, treated or ameliorated by vascular inhibition (e.g., blocking angiogenesis by vascular inhibition), a condition that is prevented, managed, treated or ameliorated by inhibiting and/or reducing the expression and/or activity of PDE4 or by inhibiting or reducing the polymerization or stability of tubulin, a cancer that is currently refractory to treatment, or a cancer that has developed resistance to multiple drugs, comprising administering to a patient in need thereof an effective amount of one or more compounds of formula I or a pharmaceutically acceptable salt, solvate or hydrate thereof.

In one embodiment, the cancer is refractory to treatment with colchicine, taxane (taxane), or vinblastine.

In one embodiment, the 3, 4-disubstituted compounds of the invention are preferred PDE4 inhibitors. In another embodiment, the 3, 4-dialkoxy substituted compounds of the present invention are preferred PDE4 inhibitors. In another embodiment, the 3, 4-dimethoxy substituted compound of the present invention is a preferred PDE4 inhibitor.

4.3.1 proliferative disorders

The compounds of the present invention and compositions comprising the compounds are useful for preventing, treating, controlling or ameliorating a proliferative disorder (e.g., cancer) or one or more symptoms thereof. Without being bound by theory, in one embodiment, the compounds of the invention bind to alpha-or beta-tubulin subunits in cancer or tumor cells and inhibit the polymerization or stability of tubulin, thereby disrupting the ability of the cancer or tumor cell to replicate. In an alternative embodiment, the compounds of the invention bind to the alpha-or beta-tubulin subunits of endothelial cells of vascularized tumors and cause a change in the shape of these cells. These changes in the shape of the endothelial cells cause the blood vessels that supply blood and oxygen to the tumor to contract, causing the tumor to shrink or die.

In one embodiment, the compounds of the invention bind to the alpha-or beta-tubulin subunit of a tumor cell or cancer cell. In another embodiment, the compounds of the invention bind to the alpha-or beta-tubulin subunit of the endothelial cells of the vascularized tumor. In a particular embodiment, the compounds of the invention are useful for preventing, controlling, treating or ameliorating cancer susceptible to tubulin binding agents. In another embodiment, the compounds of the invention are useful for preventing, controlling, treating or ameliorating cancer resistant to tubulin binding agents.

In another embodiment, the invention provides a method of inhibiting the proliferation of a cancer cell or tumor cell, comprising contacting the cancer cell or tumor cell with an effective amount of a compound of the invention. In one embodiment, the cancer cell or tumor cell is resistant to conventional cancer therapy. In another embodiment, the cancer cell or tumor cell is a multi-drug resistant cancer cell or tumor cell.

The present invention provides methods of preventing, treating, controlling or ameliorating one or more symptoms of a non-cancerous condition accompanied by cellular hyperproliferation, particularly epithelial cell hyperproliferation (e.g., in asthma, COPD, pulmonary fibrosis, bronchial hyperresponsiveness, psoriasis, lymphoproliferative disorders, and seborrheic dermatitis) and endothelial cell hyperproliferation (e.g., in restenosis, hyperproliferative vascular disease, behcet's syndrome, atherosclerosis, and macular degeneration), comprising administering one or more compounds of the present invention to an individual in need thereof. The invention also provides methods of preventing, treating, controlling or ameliorating a non-cancerous condition associated with cell hyperproliferation, the method comprising administering to an individual in need thereof one or more compounds of the invention and one or more other treatments (e.g., one or more other prophylactic or therapeutic agents) useful in preventing, treating, controlling or ameliorating the condition.

In a particular embodiment, the invention provides a method of preventing, managing, treating or ameliorating a non-cancerous condition or one or more symptoms thereof associated with cellular hyperproliferation (e.g., behcet's syndrome, sarcoidosis, keloids, pulmonary fibrosis, macular degeneration and renal fibrosis), comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of one or more compounds of the invention. In another embodiment, the invention provides a method of preventing, managing, treating, or ameliorating a non-cancerous condition or one or more symptoms thereof associated with cellular hyperproliferation (e.g., behcet's syndrome, sarcoidosis, keloids, pulmonary fibrosis, and renal fibrosis), the method comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of one or more compounds of the invention and a prophylactically or therapeutically effective amount of one or more other therapies (e.g., one or more prophylactic or therapeutic agents).

The invention includes a method of preventing, treating, controlling or ameliorating one or more symptoms of a disorder associated with cellular hyperproliferation in a subject refractory to conventional treatment of such disorders, comprising administering to the subject a prophylactically or therapeutically effective amount of one or more compounds of the invention. The invention also provides methods of preventing, managing, treating or ameliorating a non-cancerous condition associated with excessive cell proliferation in an individual who is refractory to conventional treatment of such conditions, comprising administering to an individual in need thereof one or more compounds of the invention and one or more other treatments (e.g., one or more other prophylactic or therapeutic agents) for preventing, treating, managing or ameliorating the condition.

The present invention provides methods of preventing, treating, managing or ameliorating cancer or one or more symptoms thereof, comprising administering to an individual in need thereof one or more compounds of the present invention. The invention also provides methods of preventing, treating, managing or ameliorating cancer, wherein one or more compounds of the invention are administered in combination with one or more other treatments (e.g., prophylactic or therapeutic agents) for preventing, treating, managing or ameliorating cancer or a secondary symptom.

In a specific embodiment, the invention provides a method of preventing, treating, managing or ameliorating cancer or one or more symptoms thereof, the method comprising administering to an individual in need thereof a prophylactically or therapeutically effective amount of one or more compounds of the invention. In another embodiment, the invention provides a method of preventing, treating, managing or ameliorating cancer or one or more symptoms thereof, the method comprising administering to an individual in need thereof a prophylactically or therapeutically effective amount of one or more compounds of the invention and a prophylactically or therapeutically effective amount of one or more treatments (e.g., one or more prophylactic or therapeutic agents) for preventing, treating, managing or ameliorating cancer or a secondary symptom (e.g., viral, bacterial or fungal infection).

The compounds of the invention are particularly useful as vascular targeting agents. Without being bound by theory, it is believed that the compounds of the present invention are potent anti-tumor agents because they are capable of blocking tumor vessels (e.g., existing ones) such that tumor cells die due to ischemia and hemorrhagic necrosis. Thus, the compounds of the invention are useful for disrupting or disrupting the vascular system of a tumor.

The compounds of the present invention are particularly effective as vascular targeting agents against the internal blood vessels of tumors and therefore can be used synergistically in combination with antineoplastic agents (e.g., antiangiogenic agents) effective against peripheral tumor cells. Without being bound by theory, the compounds of the present invention are particularly effective on tumor cells away from the site of vascular drug penetration failure due to their ability to target the tumor cell vasculature. Such tumor cells are more likely to be resistant to radiation and drug therapy. Thus, the compounds of the present invention are particularly effective against tumors and tumor cells that have been resistant to conventional cancer treatments.

In one embodiment, the invention provides a method of targeting, blocking or disrupting the function of tumor vasculature, comprising contacting a tumor with an effective amount of a compound of the invention.

In another embodiment, the invention provides a method of targeting, blocking or destroying the vascular endothelium of a tumor comprising contacting the tumor with an effective amount of a compound of the invention.

In another embodiment, the invention provides a method of occluding existing tumor vessels comprising contacting a tumor with an effective amount of a compound of the invention.

In another embodiment, the invention provides a method of killing a tumor cell comprising contacting the tumor cell with an effective amount of a compound of the invention.

In another embodiment, the invention provides a method of producing acute vessel collapse in a tumor cell, comprising contacting a tumor cell with an effective amount of a compound of the invention.

In another embodiment, the invention provides a method of blocking angiogenesis by vascular inhibition, the method comprising contacting a cell with an effective amount of a compound of the invention.

In another embodiment, the present invention provides a method of inhibiting tumor growth by angiogenesis, comprising administering to an individual in need thereof an effective amount of a compound of the present invention.

Without being bound by theory, it is believed that the compounds of the present invention are particularly effective at eradicating most tumors, and in one embodiment completely eradicating tumors, as they possess both vascular targeting activity (particularly effective against central tumor cells) and anti-angiogenic activity (particularly effective against peripheral tumor cells). Thus, the compounds of the present invention are particularly effective against tumors due to the synergistic effect of dual activities as a vascular targeting agent and an anti-angiogenic agent.

The compounds of the invention may be used in vitro or ex vivo for the control, treatment or amelioration of certain cancers (including but not limited to leukemia and lymphoma), such treatment including autologous stem cell transplantation. This may include a multi-step process in which an individual's autologous hematopoietic stem cells are harvested and purged of all cancer cells, followed by the administration of a high dose of a compound of the invention with or without high dose radiation therapy to eradicate the remaining bone marrow cell population of the individual, and the stem cell transplant is returned to the individual. Support care is then provided when bone marrow function is restored and the individual is rehabilitated.

One or more compounds of the invention may be used as a first, second, third, fourth or fifth line cancer therapy. The present invention provides methods of preventing, treating, managing or ameliorating cancer or one or more symptoms thereof in an individual who is refractory to conventional treatment of such cancer, comprising administering to the individual a prophylactically or therapeutically effective amount of one or more compounds of the present invention. A cancer can be determined to be refractory to treatment when at least some significant portion of the cancer cells have not been killed or their cell division ceases to respond to treatment. Such a determination can be made in vivo or in vitro by any method known in the art for assessing the effectiveness of a cancer cell therapy, and is used herein in the art-accepted sense of "incurable". In a particular embodiment, the cancer is refractory when the number of cancer cells is not significantly reduced or increased after treatment.

The present invention provides methods of preventing, managing, treating, or ameliorating cancer or one or more symptoms thereof in an individual who is refractory to existing single agent treatments for such cancer, comprising administering to the individual a prophylactically or therapeutically effective amount of one or more compounds of the present invention and a prophylactically or therapeutically effective amount of one or more treatments (e.g., one or more prophylactic or therapeutic agents) for preventing, treating, managing, or ameliorating cancer or a secondary symptom. The invention also provides methods of preventing, treating, managing or ameliorating cancer or a secondary symptom by administering one or more compounds of the invention in combination with any other therapy (e.g., radiation therapy, chemotherapy or surgery) to an individual who has proven refractory to the other therapy but not refractory to such therapy.

In a particular embodiment, the present invention provides a method of preventing, managing, treating or ameliorating cancer (refractory to colchicine, paclitaxel, docetaxel, and/or vinblastine and/or other vinblastines) or one or more symptoms thereof, said method comprising administering to an individual in need thereof a prophylactically or therapeutically effective amount of one or more compounds of the present invention. In another embodiment, the present invention provides a method of preventing, managing, treating or ameliorating cancer (refractory to cure with colchicine, paclitaxel, docetaxel and/or vinblastine) or one or more symptoms thereof, said method comprising administering to an individual in need thereof a prophylactically or therapeutically effective amount of one or more compounds of the present invention and a prophylactically or therapeutically effective amount of one or more other treatments. Other treatments according to the present invention may be chemotherapeutic agents, immunomodulators, anti-angiogenic agents, radiation therapy or surgery.

The present invention provides methods for preventing, treating, managing or ameliorating a patient having cancer and having immunosuppression due to previously received additional cancer therapy. The present invention also provides methods of preventing, treating, managing or ameliorating cancer in which chemotherapy, radiation therapy, hormonal therapy and/or biotherapeutic/immunotherapy have proven or can prove to be too toxic, i.e., produce unacceptable or intolerable side effects on the individual receiving the therapy. In addition, the invention provides methods of preventing cancer recurrence by administering one or more compounds of the invention to an individual who has received treatment and does not have any disease activity.

Cancers that may be prevented, controlled, treated or ameliorated by the methods of the invention include, but are not limited to, neoplasms, tumors (malignant and benign), and metastases, or any disease or disorder characterized by uncontrolled cell growth. The cancer may be a primary or metastatic cancer. Specific examples of cancers that may be prevented, controlled, treated or ameliorated according to the methods of the invention include, but are not limited to, cancers at the following sites: head, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon, rectum, stomach, prostate, breast, ovary, kidney, liver, pancreas, and brain. Other cancers include, but are not limited to: leukemias (such as, but not limited to, acute leukemias, acute lymphoblastic leukemias, acute myelocytic leukemias such as medulloblastoma, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, and myelodysplastic syndromes, chronic leukemias such as, but not limited to, chronic myelogenous (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia, Osler's disease, lymphomas such as, but not limited to, Hodgkin's disease, non-Hodgkin's disease, multiple myelomas such as, but not limited to, asymptomatic multiple myeloma, nonsecretory myeloma, sclerosing myeloma, plasmacytic leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; undetermined monoclonal gammopathy; benign monoclonal gammopathy; heavy chain disease; skeletal and connective tissue sarcomas, such as, but not limited to, skeletal sarcoma, osteosarcoma, chondrosarcoma, ewing's sarcoma, malignant giant cell tumor, skeletal fibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (angiosarcoma), fibrosarcoma, kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as, but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-glioma, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pinealoma, pinealoblastoma, primary brain lymphoma; breast cancers include, but are not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, paget's disease, and inflammatory breast cancer; adrenal cancer such as, but not limited to, pheochromocytoma and adrenal cortical cancer; thyroid cancers such as, but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancers such as, but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as, but not limited to, cushing's syndrome, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancers such as, but not limited to, ocular melanoma such as iris melanoma, choroidal melanoma, ciliary body melanoma, and retinoblastoma; coleopteran cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vaginal cancers such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and paget's disease; cervical cancers such as but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancers such as, but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancers such as, but not limited to, ovarian epithelial cancer, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers such as, but not limited to, squamous carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat (small cell) cell carcinoma; gastric cancers such as, but not limited to, adenocarcinoma, mycosis (polypoid), ulcer, surface spread, diffuse spread, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancer such as adenocarcinoma; bile duct cancers such as, but not limited to, papillary, nodular, and diffuse cancers; lung cancer such as non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer; testicular cancers such as, but not limited to, reproductive tumors, seminoma, degenerative, classical (typical) sperm cell non-seminoma, embryonic carcinoma, teratoma, choriocarcinoma (yolk sac tumor); prostate cancers such as, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancer; oral cancer such as, but not limited to, squamous cell carcinoma; basal carcinoma; salivary gland cancers such as, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenocystic carcinoma; pharyngeal cancers such as, but not limited to, squamous cell carcinoma and warts; skin cancers such as, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial disseminated melanoma, nodular melanoma, malignant melanoma of lentigo, acromegaly melanoma; renal cancers such as, but not limited to, renal cell carcinoma, adenocarcinoma, suprarenal adenoid tumor, fibrosarcoma, transitional cell carcinoma (renal pelvis and/or uterer); nephroblastoma; bladder cancer such as, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteosarcoma, endotheliosarcoma, lymphangioendothelioma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinoma (reviews on such conditions see Fishman et al, 1985, Medicine, second edition, J.B.Lippincott Co., Philadelphia and Murphy et al, 1997, informational Decisions: The Complete Book of cerdianosis, Treatment, and Recovery, Vig king pengan, Penguin Books U.S.A., Inc., United States of America). It is also contemplated that cancers caused by aberrations in apoptosis may also be treated using the methods and compositions of the present invention. Such cancers may include, but are not limited to, follicular lymphoma, p53 mutant cancers, hormone-dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndrome.

In a particular embodiment, the cancer prevented, controlled, treated or ameliorated according to the methods of the invention is prostate cancer, breast cancer, bone cancer, melanoma, lung cancer and ovarian cancer. In another embodiment, the cancer that is prevented, controlled, treated or ameliorated according to the methods of the invention is a metastatic tumor, including but not limited to a tumor that has or is likely to metastasize to bone (non-limiting examples are prostate, breast and lung cancers that have metastasized or are likely to metastasize to bone), a tumor that has or is likely to metastasize to lung, a tumor that has or is likely to metastasize to brain, a tumor that has or is likely to metastasize to other organs or tissues of the subject. In another embodiment, the cancer prevented, controlled, treated or ameliorated according to the methods of the invention is not associated with the expression and/or activity of TNF- α.

4.3.2 inflammatory disorders

One or more compounds of the present invention and compositions comprising the compounds are useful for preventing, treating, controlling or ameliorating an inflammatory disorder or one or more symptoms thereof. The compounds of the present invention or compositions comprising the compounds may also be administered in combination with one or more other therapies (e.g., one or more other prophylactic or therapeutic agents) for the prevention, treatment, control, or amelioration of an inflammatory disorder or one or more symptoms thereof.

In a specific embodiment, the invention provides a method of preventing, treating, managing or ameliorating an inflammatory disorder or one or more symptoms thereof, the method comprising administering to an individual in need thereof a prophylactically or therapeutically effective amount of one or more compounds of the invention. In another embodiment, the invention provides a method of preventing, treating, managing or ameliorating an inflammatory disorder or one or more symptoms thereof, the method comprising administering to an individual in need thereof a prophylactically or therapeutically effective amount of one or more compounds of the invention and a prophylactically or therapeutically effective amount of one or more other treatments (e.g., one or more other prophylactic or therapeutic agents).

The present invention provides methods for preventing, managing, treating, or ameliorating an inflammatory disorder or one or more symptoms thereof in an individual who is treated conventionally with such inflammatory disorders (e.g., methotrexate and TNF-alpha antagonists (e.g., REMICADE)^TMOr ENBREL^TM) Is refractory, said method comprising administering to said individual a prophylactically or therapeutically effective amount of one or more compounds of the invention. The invention also provides a method of preventing, treating, controlling or ameliorating an inflammatory disorder or one or more symptoms thereof in an individual who is refractory to treatment with an existing single formulation of such inflammatory disorder, the method comprising administering to the individual a prophylactically or therapeutically effective amount of one or more compounds of the invention and a prophylactically or therapeutically effective amount of one or more other treatments (e.g., one or more other prophylactic or therapeutic agents). The invention also provides methods for preventing, treating, controlling or ameliorating an inflammatory condition by administering one or more compounds of the invention in combination with any other treatment to a patient who has proven refractory to the other treatment but is not refractory to such treatment. The invention also provides alternative methods of preventing, treating, managing or ameliorating an inflammatory disorder, wherein another treatment has proven or may prove to be too toxic, i.e., produces unacceptable or intolerable side effects to the subject being treated. In addition, the present invention provides methods of preventing the recurrence of an inflammatory disorder by administering one or more compounds of the present invention to a patient who has received treatment and does not have any disease activity.

Examples of inflammatory disorders that may be prevented, controlled, treated or ameliorated with the methods of the invention include, but are not limited to, asthma, allergic disorders, inflammatory disorders characterized by type 1 mediated inflammation, inflammatory disorders characterized by type 2 mediated inflammation, fibrotic diseases (e.g., pulmonary fibrosis), psoriasis, multiple sclerosis, systemic lupus erythematosus, Chronic Obstructive Pulmonary Disease (COPD), encephalitis (encephititis), inflammatory bowel diseases (e.g., crohn's disease and ulcerative colitis), ischemic reperfusion injury, gout, behcet's syndrome, septic shock, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, rheumatoid arthritis (juvenile and adult), osteoarthritis, psoriatic arthritis, inflammatory osteolysis, sepsis, meningitis and chronic inflammation caused by chronic viral or bacterial infections. In a particular embodiment, the inflammatory disorder prevented, treated, controlled or ameliorated according to the methods of the invention is an inflammatory disorder characterized by type 2-mediated inflammation. Type 2 mediated inflammation is characterized by infiltration of eosinophilic and basophilic tissue and/or extensive mast cell degranulation, a process that relies on cross-linking of surface-bound IgE. In another embodiment, the inflammatory disorder prevented, treated, controlled or ameliorated according to the methods of the invention is asthma, Behcet's syndrome, arthritis, Chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis, renal fibrosis, gout, or an allergic disorder.

In a specific embodiment, an effective amount of one or more compounds of the invention is administered to an individual in combination with an effective amount of one or more treatments (e.g., prophylactic or therapeutic formulations) for preventing, treating, controlling or ameliorating asthma or one or more symptoms thereof. Non-limiting examples of such treatments include, but are not limited to, adrenergic agonists (e.g., catecholamines (e.g., epinephrine, isoproterenol, and isoproterenol), resorcinol (e.g., metaproterenol, terbutaline, and fenoterol), salicyclols (e.g., salbutamol)), anticholinergics (e.g., atropine sulfate, atropine methyl nitrate, and ipratropium bromide (atroment^TM) Beta 2-agonists (e.g. abuterol (VENTOLIN)), and pharmaceutically acceptable salts thereof^TMAnd PROVENTIL^TM)、Bitolterol (Tornalate)^TM) Levosalbutamol (XOPONEX)^TM) Oxinarine (ALUPENT)^TM) Pirbuterol (MAXAIR)^TM)、terbutlaine(BRETHAIRE^TMAnd BRETHINE^TM) Salbutamol (PROVENTIL)^TM、REPETABS^TMAnd VOLMAX^TM) Formoterol (FORADIL AEROLIZER)^TM) And Salmeterol (SEREVENT)^TMAnd SEREVENT DISKUS^TM) Corticosteroid (e.g., Methylprednisolone (MEDROL)^TM) Prednisone (PREDNI song)^TMAnd Deltasine^TM) And Prednisolone (PRELONE)^TM、PEDIAPRED^TM) Glucocorticoids (e.g., oral steroids or other systemic or oral steroids, inhaled glucocorticoids), other steroids, immunosuppressive agents (e.g., methotrexate and gold salts), leukotriene modifiers (e.g., montelukast (SINGULAIR) ^TM) Zafirlukast (ACCOLATE)^TM) And Zileuton (ZYFLO)^TM) Mast cell stabilizers (e.g., cromolyn sodium (INTAL))^TM) And sodium naproxen sodium (tilode)^TM) Methyl xanthines (e.g. theophylline (UNIPHYL)^TM、THEO-DUR^TM、SLO-BID^TM、AND TEHO-42^TM) And mucolytic agents (e.g., acetylcysteine).

In a specific embodiment, an effective amount of one or more compounds of the invention is administered to an individual in combination with an effective amount of one or more treatments (e.g., prophylactic or therapeutic formulations) for preventing, treating, controlling or ameliorating allergy or one or more symptoms thereof. Non-limiting examples of such treatments include anti-mediator agents (e.g., antihistamines, see table 3), corticosteroids, decongestants, sympathomimetics (e.g., alpha-adrenergic and beta-adrenergic), theophylline and its derivatives, glucocorticoids, and immunotherapy (e.g., repeated long-term injections of allergens, short-term desensitization, and toxicant immunotherapy).

TABLE 3-H₁Antihistaminic agents

Chemical Classification and representative drugs	Conventional daily dosage
		Ethanolamine Diphenamine (Diphehydramine) chloromalastine ethylenediamine tripheniramine alkylamine brompheniramine chlorpheniramine triprolidine (1.25mg/5ml) phenothiazine promethazine piperazine hydroxyzine piperidine astemizole (non-sedative) azatadine cetirizine cyproheptadine	25-50mg every 4-6 hours, 0.34-2.68mg every 12 hours, 25-50mg every 4-6 hours, 4mg every 4-6 hours; or 8-12mg of the sustained release preparation every 8-12 hours and 4mg every 4-6 hours; or 8-12mg per 8-12 hr slow release dosage form 2.5mg per 4-6 hr before sleep 25mg per 6-8 hr 25mg10mg/d per 12 hr 1-2mg10mg/d per 6-8 hr 4mg per 12 hr 60mg

Fexofenadine (non-sedating) loratadine (Loratidine) (non-sedating)

10mg per 24 hours

In a particular embodiment, an effective amount of one or more compounds of the invention is administered to an individual in combination with an effective amount of one or more treatments (e.g., prophylactic or therapeutic formulations) for preventing, treating, controlling or ameliorating COPD or one or more symptoms thereof. Non-limiting examples of such treatments include, but are not limited to, bronchodilators (e.g., short acting beta)₂Adrenergic agonists (e.g. salbutamol, pirbuterol, terbutaline and metaproterenol), long-acting beta₂Adrenergic agonists (e.g. oral sustained release salbutamol and inhaled salmeterol), anticholinergics (e.g. ipratropium bromide) and theophylline and its derivatives (therapeutic theophylline doses preferably 10-20ug/mL)), glucocorticoids, exogenous alpha-agonists₁AT (e.g., alpha derived from pooled human plasma AT a weekly 60mg/kg dose ₁AT), oxygen, lung transplant, lung volume reduction, tracheal intubation, ventilatory support, annual vaccination with influenza vaccine and vaccination with 23-valent polysaccharide pneumococcus, exercise and smoking cessation.

In a specific embodiment, an effective amount of one or more compounds of the invention is administered to an individual in combination with an effective amount of one or more treatments (e.g., prophylactic or therapeutic formulations) for preventing, treating, controlling or ameliorating pulmonary fibrosis or one or more symptoms thereof. Non-limiting examples of such treatments include oxygen, corticosteroids (e.g., prednisone given daily, starting at 1-1.5mg/kg/d (up to 100mg/d), administered for 6 weeks, slowly decreasing to a minimum maintenance dose of 0.25mg/kg/d over 3-6 months), cytotoxic drugs (e.g., cyclophosphamide, orally administered 100mg once a day, and azathioprine orally administered 3mg/kg, up to 200mg once a day), bronchodilators (e.g., short and long acting beta agonists), and the like₂Adrenergic agonists, anticholinergics and theophyllines and their derivatives) and antihistamines (e.g. diphenhydramine and doxylamine).

Anti-inflammatory therapies and their dosages, routes of administration, and recommended use are known in the art and have been described in, for example, the Physician's Desk Reference (57 th edition, 2003).

4.3.3 central nervous System disorders

One or more compounds of the present invention and compositions comprising such compounds are useful for preventing, treating, controlling or ameliorating a central nervous system disorder or one or more symptoms thereof. The compounds of the present invention or compositions comprising the compounds may also be administered in combination with one or more other therapies (e.g., one or more other prophylactic or therapeutic agents) for the prevention, treatment, control, or amelioration of a central nervous system disorder or one or more symptoms thereof.

Central nervous system disorders include, but are not limited to, parkinson's disease, bradykinesia, myotonia, parkinsonian tremor, parkinsonian gait disorder, motor freezing, depression, long-term memory loss, rubinstein-tettiy syndrome (RTS), dementia, sleep disorders, postural instability, hypokinesia, inflammation, synuclein disorders, multiple system atrophy (multiple system atrophies), striatal substantia nigra degeneration (striato-cerebellar atrophy), olivopontocerebellar atrophy, Shy-drager syndrome, motor neuron disease with parkinsonism characteristics, Lewy body dementia (Lewy body dementia), Tau pathological conditions, progressive supranuclear palsy (progressive supranuclear palsy), corticobasal degeneration (corticobasal degeneration), frontotemporal dementia (frontotemporal dementia), amyloid pathological conditions, mild cognitive dysfunction, alzheimer's disease, parkinson's disease, motor disorder, motor freezing, depression, long-term memory loss, multiple system memory loss, motor impairment, alzheimer's disease with parkinsonism, genetic disorders which may be characteristic of Parkinson's disease, Wilson's disease, Hallervorden-Spatz disease, Chediak-Hagashi disease, SCA-3 spinocerebellar ataxia, X-desmoplastic Parkinson's syndrome, Huntington's disease, prion diseases, hyperkinesia, chorea, ballism, dystonic tremor, Amyotrophic Lateral Sclerosis (ALS), central nervous system trauma, and myoclonus.

In a particular embodiment of the invention, the compounds of the invention are used, administered or formulated with one or more second active ingredients to treat, prevent or manage central nervous system disorders. Examples of second active ingredients include, but are not limited to, dopamine agonists, levodopa, compounds used to enhance levodopa therapy such as monoamine oxidase inhibitors (MAOs) and catechol-o-methyltransferase inhibitors (COMTs), amantadine, anticholinergics, antiemetics, and other standard treatments for central nervous system disorders. In another example, the second active ingredient is an anti-inflammatory agent including, but not limited to, non-steroidal anti-inflammatory drugs (NSAIDs), methotrexate, leflunomide, antimalarials and sulfasalazine, gold salts, glucocorticoids, immunosuppressive agents and other standard treatments for central nervous system disorders.

4.4 compositions and methods of administering treatments

The present invention provides compositions for treating, preventing and ameliorating disorders characterized by or accompanied by aberrant angiogenesis, proliferative disorders, inflammatory disorders, and disorders prevented, controlled, treated or ameliorated by inhibiting or reducing the expression and/or activity of PDE4 or by inhibiting or reducing the polymerization or stability of tubulin. In a particular embodiment, the composition comprises one or more compounds of the present invention or a pharmaceutically acceptable salt, solvate or hydrate thereof. In another embodiment, the compositions of the invention comprise one or more prophylactic or therapeutic agents other than a compound of the invention, or a pharmaceutically acceptable salt, solvate or hydrate thereof, that are known to be useful, or that have been used, or are currently being used, to prevent, treat, control or ameliorate a condition (e.g., a condition characterized by or accompanied by aberrant angiogenesis, a proliferative condition, an inflammatory condition, or a condition that is prevented, controlled, treated or ameliorated by inhibition of PDE4, or by reduction or inhibition of the polymerization or stability of tubulin), or one or more symptoms thereof. In another embodiment, the compositions of the invention comprise one or more compounds of the invention, or a pharmaceutically acceptable salt, solvate or hydrate thereof, and one or more prophylactic or therapeutic agents known to be useful, or already in use, or currently in use, for preventing, treating, managing or ameliorating a disorder (e.g., a disorder characterized by or accompanied by aberrant angiogenesis, a proliferative disorder, an inflammatory disorder, or a disorder which is prevented, controlled, treated or ameliorated by inhibition of PDE4, or by reduction or inhibition of polymerization or stability of tubulin) or one or more symptoms thereof.

In a specific embodiment, the composition comprises one or more compounds of the present invention or a pharmaceutically acceptable salt, solvate or hydrate thereof and one or more immunomodulators. In another embodiment, a composition comprises one or more compounds of the present invention or a pharmaceutically acceptable salt, solvate or hydrate thereof and one or more anti-angiogenic agents, wherein the anti-angiogenic agent is not a compound of the present invention. In another embodiment, a composition comprises one or more compounds of the present invention, or a pharmaceutically acceptable salt, solvate or hydrate thereof, and one or more anti-inflammatory agents, wherein the anti-inflammatory agent is not a compound of the present invention. In another embodiment, a composition comprises one or more compounds of the present invention, or a pharmaceutically acceptable salt, solvate or hydrate thereof, and one or more anti-cancer agents, wherein the anti-cancer agents are not compounds of the present invention. According to this embodiment, the anti-cancer agent may or may not be an immunomodulatory agent or an anti-angiogenic agent. In another embodiment, a composition comprises one or more compounds of the present invention, or a pharmaceutically acceptable salt, solvate or hydrate thereof, and one or more antiviral agents. In another embodiment, the composition comprises one or more compounds of the present invention or a pharmaceutically acceptable salt, solvate or hydrate thereof or one or more antibiotics. In another embodiment, a composition comprises one or more compounds of the invention, or a pharmaceutically acceptable salt, solvate or hydrate thereof, and any combination of one, two, three or more of the following prophylactic or therapeutic agents: an immunomodulator, an anti-angiogenic agent, an anti-cancer agent other than an immunomodulator or an anti-angiogenic agent, an anti-inflammatory agent, an anti-viral agent or an antibacterial agent (e.g., an antibiotic).

In a preferred embodiment, the composition of the invention is a pharmaceutical composition or a separate unit dosage form. The pharmaceutical compositions and individual unit dosage forms of the invention comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., a compound of the invention or other prophylactic or therapeutic agent) and, typically, one or more pharmaceutically acceptable carriers or excipients. In a particular embodiment and herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., freund's adjuvant (complete and incomplete)), excipient, or vehicle used to administer the treatment. Such pharmaceutical carriers can be sterile liquids, such as water and oils (including those of petroleum, animal, vegetable or synthetic origin), for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in "Remington's pharmaceutical science" by e.w. martin.

Typical pharmaceutical compositions and dosage forms contain one or more excipients. Suitable excipients are well known to those skilled in the art of pharmacy, and non-limiting examples of suitable excipients include 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. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors known in the art, including, but not limited to, the manner in which the dosage form is administered to a patient and the particular active ingredient in the dosage form. The compositions or individual unit dosage forms may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, if desired.

Lactose-free compositions of the invention may comprise excipients well known in the art and listed, for example, in the United States Pharmacopeia (USP) SP (XXI)/NF (XVI). Generally, lactose-free compositions comprise an active ingredient, a binder/filler, a pharmaceutically compatible and pharmaceutically acceptable amount of a lubricant. Preferred lactose-free dosage forms comprise the active ingredient, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

Since water promotes the breakdown of some compounds, the present invention also includes anhydrous pharmaceutical compositions and dosage forms comprising active ingredients. For example, it is generally accepted in pharmaceutical technology that adding water (e.g., 5%) is a method to simulate long-term storage to determine attributes of a formulation over time such as shelf life or stability. See, for example, (Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, NY, NY, 1995, pages 379-80). In fact, water and heat accelerate the decomposition of some compounds. Thus, water can have a significant impact on the formulation because of the moisture and/or humidity commonly encountered in the manufacture, handling, packaging, storage, transportation and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared by using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms of the invention comprising lactose and at least one active ingredient comprising a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or humidity during manufacture, packaging and/or storage is expected.

Anhydrous pharmaceutical compositions are prepared and stored to maintain their anhydrous nature. Accordingly, anhydrous compositions are preferably packaged with materials known to prevent exposure to water so that they can be packaged in a suitable formulation box. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

The invention also includes pharmaceutical compositions and dosage forms comprising one or more compounds that reduce the rate of decomposition of an active ingredient. Such compounds (referred to herein as "stabilizers") include, but are not limited to, antioxidants (e.g., ascorbic acid), pH buffers, or salt buffers.

The pharmaceutical compositions and discrete unit dosage forms may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such compositions and dosage forms will comprise a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent (preferably in pure form) and an appropriate amount of carrier to provide an appropriate dosage form for administration to a patient. The formulation should be adapted to the mode of administration. In a preferred embodiment, the pharmaceutical composition or separate unit dosage form is sterile and in a form suitable for administration to an individual, preferably an animal individual, more preferably a mammalian individual, most preferably a human individual.

The pharmaceutical compositions of the present invention are formulated to accommodate their intended route of administration. Examples of routes of administration include, but are not limited to, parenteral (e.g., intravenous), subdermal, subcutaneous, oral (e.g., inhalation), intranasal, transdermal (topical), transmucosal, intratumoral, intrasynovial, and rectal administration. In a specific embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to a human. In a preferred embodiment, the pharmaceutical composition is formulated for subcutaneous administration to a human according to conventional procedures. Typically, compositions for intravenous administration are sterile isotonic aqueous buffer solutions. If desired, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine (lignocane), to reduce pain at the site of injection. Examples of dosage forms include, but are not limited to: tablets, caplets, capsules (e.g., soft elastic gelatin capsules), cachets, lozenges, dispersions, suppositories, ointments, poultices (poultices), pastes, powders, dressings, creams, plasters, solutions, patches, aerosols (e.g., nasal sprays or inhalants), gels, liquid dosage forms suitable for oral or mucosal administration to a subject, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs, liquid dosage forms suitable for parenteral administration to a patient, and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

The composition, shape and type of dosage forms of the present invention generally vary depending on their use. For example, a dosage form for the acute treatment of inflammation or related disorders may comprise a greater amount of one or more active ingredients than a dosage form for the chronic treatment of the same disorder. Likewise, the therapeutically effective dosage form may vary depending on the type of cancer. Similarly, parenteral dosage forms may contain smaller amounts of one or more active ingredients than oral dosage forms in treating the same disease or condition. These and other aspects of the particular dosage forms encompassed by the present invention will vary from one another and are well known to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18 th edition, MackPublishing, Easton PA, 1990.

Generally, the ingredients of the compositions of the present invention are provided in unit dosage forms, either individually or in admixture, such as a lyophilized powder or anhydrous concentrate in an air-tight container, e.g., an ampoule or sachet indicating the amount of active ingredient. If the composition is administered by infusion, the composition may be formulated in an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is administered by injection, sterile water for injection or saline ampoules may be provided to allow the components to be mixed prior to administration. A typical dosage form of the invention comprising a compound of the invention or a pharmaceutically acceptable salt, solvate or hydrate thereof is administered in a range of from about 1mg to about 1000mg per day, in a single dose once a day in the morning, but preferably in divided doses throughout the day with meals.

4.4.1 oral dosage forms

Pharmaceutical compositions of the present invention suitable for oral administration may be in discrete dosage forms such as, but not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms comprise a predetermined amount of active ingredient and may be prepared by pharmaceutical methods well known to those skilled in the art. See Remington's Pharmaceutical Sciences, 18 th edition, MackPublishing, Easton PA, 1990.

Typical oral dosage forms of the invention are prepared by intimately mixing the active ingredient with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients may take a variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.

Tablets and capsules using solid excipients represent the most advantageous oral dosage form due to the ease of administration. If desired, the tablets may be coated by standard aqueous or non-aqueous techniques. Such dosage forms may be prepared by any pharmaceutical method. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredient with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation, if necessary.

For example, tablets may be prepared by compression or molding. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with excipients. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Examples of excipients that may be used in the oral dosage forms of the present invention include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums (e.g., acacia), sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, carboxymethylcellulose sodium), polyvinylpyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropylmethylcellulose (e.g., nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof. The binder or filler in the pharmaceutical compositions of the present invention typically comprises from about 50% to about 99% by weight of the pharmaceutical composition or dosage form.

Suitable forms of microcrystalline cellulose include, but are not limited to, materials available under the trade names AVICEL-PH-101, AVICEL-PH-103, AVICEL RC-581, AVICEL-PH-105 (available from FMC corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. One specific binder is a mixture of microcrystalline cellulose and sodium carboxymethylcellulose sold under the trade name AVICEL RC-581. Suitable anhydrous or low water content excipients or additives include AVICEL-PH-103^TMAnd Starch 1500 LM.

Disintegrants are used in the compositions of the invention to allow the tablets to disintegrate when exposed to an aqueous environment. Tablets containing too much disintegrant may disintegrate during storage, while tablets with too little disintegrant may not disintegrate at the required rate or under the required conditions. Thus, a sufficient amount of disintegrant, neither too much nor too little, should be used to prepare the solid oral dosage form of the present invention so as not to adversely alter the release of the active ingredient. The amount of disintegrant used varies with the type of formulation and is known to those skilled in the art. Typical pharmaceutical compositions comprise from about 0.5% to about 15% by weight of disintegrant, specifically from about 1% to about 5% by weight of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, cross-linked polyvinylpyrrolidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pregelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants useful in the pharmaceutical compositions and dosage forms of the present invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerol, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof. Other lubricants include, for example, syloid silica gel (AEROSIL 200, manufactured by w.r.grace co.baltimore, MD), condensation aerosol of synthetic silica (sold by Degussa co.plano, TX), CAB-O-SIL (fumed silica product, sold by Cabot co.boston, MA), and mixtures thereof. If used, lubricants are generally used in amounts less than about 1% by weight of the pharmaceutical composition or dosage form in which they are used.

4.4.2 sustained Release dosage forms

The active ingredients of the present invention can be administered by controlled release methods or administration devices well known to those skilled in the art. Examples include, but are not limited to, examples as described in U.S. Pat. nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which is incorporated herein by reference. Such dosage forms may be used to provide slow and controlled release of one or more active ingredients by using hydroxypropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof to provide the desired release profile in varying proportions. Suitable controlled release formulations known to those skilled in the art include those described herein and can be readily selected for use with the active ingredients of the present invention. The invention thus encompasses individual unit dosage forms suitable for controlled release and for oral administration, such as, but not limited to, tablets, capsules, gelatin capsules and caplets.

The common goal of all controlled release pharmaceutical products is to improve drug therapy over their non-controlled release counterparts. Ideally, the use of an optimally designed controlled release formulation in drug therapy is characterized by the use of a minimum amount of drug substance in a minimum amount of time to cure or control the condition. Advantages of controlled release formulations include prolonged activity of the drug, reduced dosage frequency, and improved patient compliance. In addition, controlled release formulations can be used to affect the onset of action or other attributes (e.g., blood levels) and thus can affect the appearance of side (adverse) effects.

Most controlled release formulations are designed to initially release a certain amount of the drug (active ingredient) to rapidly produce the desired therapeutic effect, and to gradually and continuously release other amounts of the drug to maintain that level of therapeutic or prophylactic effect over an extended period of time. To maintain such constant levels of drug in the body, the drug must be released from the dosage form at a rate that replaces the amount of drug that is metabolized and excreted. Controlled release of an active ingredient can be facilitated by a variety of conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.

4.4.3 parenteral dosage forms

Parenteral dosage forms can be administered to an individual by a variety of routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and arterial. Since their administration typically circumvents the patient's natural defense barrier against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to the patient. Examples of parenteral dosage forms include, but are not limited to, solutions for injection, dry products that can be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions for injection, and emulsions.

Suitable vehicles that can be used to provide the parenteral dosage forms of the invention are well known to those skilled in the art. Examples include, but are not limited to: water for injection USP, aqueous vehicles (such as, but not limited to, sodium chloride injection, ringer's injection, dextrose and sodium chloride injection, and lactated ringer's injection), water miscible vehicles (such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol), and non-aqueous vehicles (such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate).

Compounds that enhance the solubility of one or more of the active ingredients disclosed herein may also be incorporated into the parenteral dosage forms of the invention.

4.4.4 transdermal, topical and mucosal dosage forms

Transdermal, topical, and mucosal dosage forms of the invention include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, gels, solutions, emulsions, suspensions, or other forms known to those skilled in the art. See Remington's Pharmaceutical Sciences, 16 th and 18 th edition, Mack Publishing, Easton PA (1980 & 1990) and Introduction to Pharmaceutical Dosage Forms, 4 th edition, Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treating mucosal tissue in the oral cavity can be formulated as mouthwashes or as mouth gels. In addition, transdermal dosage forms include "depot" or "matrix" patches that are applied to the skin and can be maintained for a specified period of time to allow penetration of a desired amount of active ingredient.

Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide the transdermal, topical and mucosal dosage forms encompassed by the present invention are well known to those skilled in the pharmaceutical arts and depend on the particular tissue to which a particular pharmaceutical composition or dosage form is to be administered. Accordingly, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, 1, 3-butylene glycol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form lotions, tinctures, creams, emulsions, gels, or ointments that are non-toxic and pharmaceutically acceptable. If desired, a warming solution or a wetting agent may be added to the pharmaceutical composition and the dosage form. Examples of such additional ingredients are well known in the art. See Remington's Pharmaceutical Sciences, 16 th and 18 th edition, MackPublishing, Easton PA (1980 & 1990).

The additional components may be administered prior to, simultaneously with, or subsequent to treatment with the active ingredients of the present invention, depending on the particular tissue being treated. For example, the permeation enhancer may aid in the delivery of the active ingredient to the tissue. Suitable penetration enhancers include, but are not limited to: acetone, various alcohols (e.g., ethanol, oleyl alcohol and tetrahydrofuryl alcohol), alkyl sulfoxides (e.g., dimethyl sulfoxide), dimethylacetamide, dimethylformamide, polyethylene glycol, pyrrolidones (e.g., polyvinylpyrrolidone), Kollidon grades (Povidone, Polyvidone), urea and various water-soluble or water-insoluble sugar esters, such as tween 80 (polysorbate 80) and span 60 (sorbitan monostearate).

The pH of the pharmaceutical composition or dosage form or the pH of the tissue to which the pharmaceutical composition or dosage form is administered may also be adjusted to improve delivery of one or more active ingredients. Similarly, the polarity of the solvent carrier, its ionic strength, or tonicity may also be adjusted to improve delivery. Compounds (e.g., stearates) can also be added to the pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients to improve delivery. In this regard, stearates can be used as lipid vehicles, emulsifiers or surfactants for formulations, as delivery or penetration enhancers. Different salts, hydrates or solvates of the active ingredients can be used to further adjust the properties of the resulting composition.

4.4.5 dosage and frequency

The amount of a compound or composition of the invention effective to prevent, treat, control or ameliorate a condition (e.g., a condition characterized by or accompanied by aberrant angiogenesis, a proliferative condition, an inflammatory condition, or a condition prevented, controlled, treated or ameliorated by inhibiting or reducing PDE4 or inhibiting or reducing tubulin polymerization or stability, or by reducing or inhibiting tubulin polymerization or stability) or one or more symptoms thereof will vary with the nature and severity of the disease or condition and the route of administration of the active ingredient. The frequency and dosage will also vary with the particular factor of each patient, depending on the particular treatment (e.g., therapeutic or prophylactic), the severity of the disease, disorder or condition being administered, the route of administration, as well as the age, body, weight, response, and past medical history of the patient. Effective doses can be extrapolated from dose-response curves obtained from in vivo or animal model test systems. One skilled in the art will select an appropriate regimen by considering such factors and in accordance with dosages reported and recommended in the literature, for example, in the physicians' Desk Reference (57 th edition, 2003).

Exemplary doses of small molecules include milligram or microgram amounts of small molecule per kilogram of individual or sample weight (e.g., about 1 microgram/kilogram to about 500 milligrams/kilogram, about 100 microgram/kilogram to about 5 milligrams/kilogram, or about 1 microgram/kilogram to about 50 micrograms/kilogram).

In general, the recommended daily dosage of the compounds of the invention for use in the conditions described herein is from about 0.01mg to about 1000mg per day in a single dose once a day, preferably in divided doses throughout the day. In one embodiment, the daily dose is administered in divided doses twice daily. Specifically, the daily dose should be from about 5mg to about 500mg per day, more specifically, from about 10mg to about 200mg per day. During the management of the patient's condition, treatment should be initiated at lower doses, perhaps from about 1mg to about 25mg, and if necessary increased to about 200mg to about 1000mg per day, administered in single or divided doses, depending on the overall response of the patient. In some cases, it may be desirable to use dosages of the active ingredient which are outside the scope of the present disclosure, as will be apparent to those skilled in the art. In addition, the clinician or attending physician will know how and when to interrupt, adjust or terminate therapy according to the individual patient's response.

Different therapeutically effective amounts may be used for different diseases or conditions, as will be appreciated by those skilled in the art. Similarly, the dosages and dose frequency schedules described above also include amounts sufficient to prevent, control, treat or ameliorate such disorders, but insufficient to cause or sufficient to reduce adverse effects associated with the compounds of the invention. Furthermore, when multiple doses of a compound of the invention are administered to a patient, not all doses need be the same. For example, the dosage administered to a patient can be increased to enhance the prophylactic or therapeutic effect of the compound, or the dosage can be decreased to reduce one or more side effects experienced by the particular patient.

In a particular embodiment, the dose of a composition of the invention or a compound of the invention administered to a patient to prevent, treat, control or ameliorate a condition (e.g., a condition characterized by or accompanied by aberrant angiogenesis, a proliferative condition, an inflammatory condition, or a condition prevented, controlled, treated or ameliorated by inhibiting PDE4, or by reducing or inhibiting tubulin polymerization or stability) or one or more symptoms thereof is 150 μ g/kg, preferably 250 μ g/kg, 500 μ g/kg, 1mg/kg, 5mg/kg, 10mg/kg, 25mg/kg, 50mg/kg, 75mg/kg, 100mg/kg, 125mg/kg, 150mg/kg or 200mg/kg or more (based on the body weight of the patient). In another embodiment, the composition of the invention or the compound of the invention administered to a subject to prevent, treat, manage or ameliorate a condition (e.g., a condition characterized by or accompanied by abnormal angiogenesis, a proliferative condition, an inflammatory condition, or a condition prevented, managed, treated or ameliorated by inhibiting PDE4, or by reducing or inhibiting tubulin polymerization or stability) or one or more symptoms thereof is administered in a unit dose of 0.1mg to 20mg, 0.1mg to 15mg, 0.1mg to 12mg, 0.1mg to 10mg, 0.1mg to 8mg, 0.1mg to 7mg, 0.1mg to 5mg, 0.1 to 2.5mg, 0.25mg to 20mg, 0.25 to 15mg, 0.25 to 12mg, 0.25 to 10mg, 0.25 to 8mg, 0.25mg to 7mg, 0.25mg to 5mg, 0.5mg to 2.5mg, 1mg to 20mg, 1mg to 12mg, 1mg to 10mg, 1mg to 8mg, 1mg to 7mg, 1mg to 5mg, 1mg to 12mg, 1mg to 7mg, 1mg, or more mg to 7mg of a compound of a, 1mg to 5mg or 1mg to 2.5 mg.

In addition to the compounds of the invention, dosages of prophylactic or therapeutic preparations that have been used, or are being used, in the prevention, treatment, control, or amelioration of a disorder (e.g., a disorder characterized by or accompanied by aberrant angiogenesis, a proliferative disorder, an inflammatory disorder, or a disorder prevented, controlled, treated, or ameliorated by inhibition of PDE4, or by reduction or inhibition of tubulin polymerization or stability), or one or more symptoms thereof, can be used in the combination therapies of the invention. Preferably, the dosage employed in the combination therapy of the invention is lower than that which has been used or is being used to prevent, treat, control or ameliorate a condition (e.g. a condition characterized by or accompanied by aberrant angiogenesis, a proliferative condition, an inflammatory condition or a condition which is prevented, controlled, treated or ameliorated by inhibition of PDE4, or by reduction or inhibition of tubulin polymerization or stability) or one or more symptoms thereof. The recommended dosage Of a formulation currently used to prevent, treat, manage or ameliorate a condition (e.g., a condition characterized by or accompanied by aberrant angiogenesis, a proliferative condition, an inflammatory condition, or a condition prevented, managed, treated or ameliorated by inhibiting PDE4, or by reducing or inhibiting tubulin polymerization or stability) or one or more symptoms thereof can be obtained from any reference in the art, including but not limited to Goodman & Gilman's pharmaceutical basic Of basic Therapeutics (pharmacological Basis Of basic therapy) edited by Hardman et al, 9 th edition, 1996, Mc-Graw-Hill, New York; physician's Desk Reference (PDR), 57 th edition, 2003, Medical Economics Co., Inc., Montvale, NJ, which are incorporated herein by Reference.

In various embodiments, the administration time interval for each treatment (e.g., prophylactic or therapeutic formulation) is less than 5 minutes, less than 30 minutes, 1 hour, about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 5 hours, about 5 hours to about 6 hours, about 6 hours to about 7 hours, about 7 hours to about 8 hours, about 8 hours to about 9 hours, about 9 hours to about 10 hours, about 10 hours to about 11 hours, about 11 hours to about 12 hours, about 12 hours to about 18 hours, 18 hours to 24 hours, 24 hours to 36 hours, 36 hours to 48 hours, 48 hours to 52 hours, 52 hours to 60 hours, 60 hours to 72 hours, 72 hours to 84 hours, 84 hours to 96 hours, or 96 hours to 120 hours. In a preferred embodiment, two or more treatments (e.g., prophylactic or therapeutic formulations) are administered to the same viewing patient.

In certain embodiments, one or more compounds of the invention and one or more other therapies (e.g., prophylactic or therapeutic agents) are administered cyclically. Cycling therapy involves administering a first treatment (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by a second treatment (e.g., a second prophylactic or therapeutic agent) for a period of time, followed by a third treatment (e.g., a third prophylactic or therapeutic agent) for a period of time, and repeating the order of administration (i.e., the cycle) to reduce the development of resistance to one of the agents, avoid or reduce side effects of one of the agents, and/or improve the effectiveness of the treatment.

In certain embodiments, the administration of the same compound of the invention may be repeated with intervals of at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. In other embodiments, the same prophylactic or therapeutic formulation may be repeatedly administered, and administration may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.

In a particular embodiment, the invention provides a method of preventing, treating, managing or ameliorating a disorder (e.g., a disorder characterized by or accompanied by abnormal angiogenesis, a proliferative disorder, an inflammatory disorder, or a disorder prevented, controlled, treated or ameliorated by inhibiting PDE4, or by reducing or inhibiting tubulin polymerization or stability) or one or more symptoms thereof, which method comprises administering a dose of at least 150 μ g/kg, preferably at least 250 μ g/kg, at least 500 μ g/kg, at least 1mg/kg, at least 5mg/kg, at least 10mg/kg, at least 25mg/kg, at least 50mg/kg, at least 75mg/kg, at least 100mg/kg, at least 125mg/kg, at least 150mg/kg or at least 200mg/kg or more of one or more of the compounds of the invention once every 3 days, Preferably, the administration is once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 10 days, once every 2 weeks, once every 3 weeks, or once a month to an individual in need thereof.

The invention provides methods of preventing, treating, managing or ameliorating a disorder (e.g., a disorder characterized by or accompanied by aberrant angiogenesis, a proliferative disorder, an inflammatory disorder, or a disorder prevented, controlled, treated or ameliorated by inhibiting PDE4, or by reducing or inhibiting tubulin polymerization or stability), or one or more symptoms thereof, comprising: (a) administering one or more doses of a prophylactically or therapeutically effective amount of one or more compounds of the invention to an individual in need thereof; and (b) monitoring the mean absolute lymphocyte count of said individual after administration of a dose of said compound of the invention. Furthermore, it is preferred that the amount of the dose is a prophylactically or therapeutically effective amount of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 parts of one or more compounds of the present invention.

In a particular embodiment, the invention provides a method of preventing, treating, managing or ameliorating a disorder (e.g., a disorder characterized by or accompanied by aberrant angiogenesis, a proliferative disorder, an inflammatory disorder, or a disorder prevented, controlled, treated or ameliorated by inhibiting PDE4, or by reducing or inhibiting tubulin polymerization or stability), or one or more symptoms thereof, comprising: (a) administering to an individual in need thereof one or more compounds of the invention at a dose of at least 150 μ g/kg, preferably at least 250 μ g/kg, at least 500 μ g/kg, at least 1mg/kg, at least 5mg/kg, at least 10mg/kg, at least 25mg/kg, at least 50mg/kg, at least 75mg/kg, at least 100mg/kg, at least 125mg/kg, at least 150mg/kg or at least 200mg/kg or more; and (b) when the average absolute lymphocyte count of said individual is at least about 500 cells/mm ³Preferably at least about 600cells/mm³At least about 700cells/mm³At least about 750cells/mm³At least about 800cells/mm³At least about 850cells/mm³Or at least about 900cells/mm³Administering one or more subsequent doses to the individual.

4.5 biological assay

The anti-cancer activity of the pharmaceutical compositions and compounds of the invention can be determined by using any suitable animal model, including but not limited to SCID mice bearing tumors or injected with malignant cells. Examples of animal models for lung cancer include, but are not limited to, the lung cancer animal model described by Zhang & Roth (1994, In Vivo 8 (5): 755-69) and the transgenic mouse model with disrupted p53 function (see, e.g., Morris et al, 1998, J La State Med Soc 150 (4): 179-85). Examples of animal models for breast cancer include, but are not limited to, Transgenic mice overexpressing cyclin D1 (see, e.g., Hosokawa et al, 2001, Transgenic Res 10 (5): 471-8). Examples of animal models for colon cancer include, but are not limited to, TCR b and p53 double knockout mice (see, e.g., Kado et al, 2001, cancer Res 61 (6): 2395-8). Examples of animal models for pancreatic cancer include, but are not limited to, a metastatic model of Panc02 mouse pancreatic cancer (see, e.g., Wang et al, 2001, Int J Pancreatol 29 (1): 37-46) and a homozygous nude mouse (nu-numic) that produces subcutaneous pancreatic tumors (see, e.g., Ghaneh et al, 2001, Gene Ther 8 (3): 199-208). Examples of animal models for non-Hodgkin's lymphoma include, but are not limited to, severe combined immunodeficiency ("SCID") mice (see, e.g., Bryant et al, 2000, Lab Invest 80 (4): 553-73) and IgHmu-HOX11 transgenic mice (see, e.g., Hough et al, 1998, Proc Natl Acad Sci USA 95 (23): 13853-8). Examples of animal models for esophageal cancer include, but are not limited to, transgenic mice for the human papillomavirus type 16E7 oncogene (see, e.g., Herber et al, 1996, J Virol 70 (3): 1873-81). Examples of animal models for rectal cancer include, but are not limited to, the Apc mouse model (see, e.g., Fodde & Smits, 2001, Trends Mol Med 7 (8): 369-73 and Kuraguchi et al, 2000, Oncogene 19 (50): 5755-63).

The anti-inflammatory activity of the pharmaceutical compositions and compounds of the present invention can be determined by a variety of experimental animal models of inflammatory Arthritis known in the art, as described in Crofford l.j. and Wilder r.l., "Arthritis and Autoimmunity in Animals", in Arthritis and Allied Conditions: a Textbook of Rheumatology, McCarty et al (eds.), Chapter 30 (Lee and Febiger, 1993). Experimental and spontaneous animal models of inflammatory arthritis and autoimmune rheumatic diseases may also be used to assess the anti-inflammatory activity of the pharmaceutical compositions and compounds of the invention. The following are exemplary evaluations provided as examples and not by way of limitation.

The major animal models of arthritis or inflammatory diseases known and widely used in the art include: rat models of adjuvant-induced Arthritis, rat and mouse models of collagen-induced Arthritis, and rat, rabbit and hamster models of antigen-induced Arthritis, all as described in Crofford l.j. and Wilder r.l., "Arthritis and Autoimmunity in Animals", in Arthritis and Allied Conditions: a Textbook of Rheumatology, McCarty et al (eds.), Chapter 30 (Lee and Febiger, 1993), which is incorporated herein by reference.

The anti-inflammatory activity of the pharmaceutical compositions and compounds of the present invention can be assessed by a rat model of carrageenan-induced arthritis. Carrageenan-induced arthritis has also been used in rabbits, dogs and pigs in studies of chronic arthritis or inflammation. Quantitative tissue morphology assessment was used to determine treatment efficacy. Methods for using Carrageenan to induce Arthritis models are described in Hansra P. et al, "Carrageenan-Induced Arthritis in the Rat", Inflammation, 24 (2): 141-155, (2000). Animal models of zymosan-induced inflammation known and described in the art are also commonly used.

The Anti-inflammatory activity of the pharmaceutical compositions and compounds of the invention can also be assessed by measuring the inhibition of Carrageenan-Induced Paw Edema in rats using a modification of the method described in Winter c.a. et al, "carrageenann-Induced Edema in Hind Paw soft tissue Drugs" Proc.Soc.exp. biol. Med.111, 544-. This assay has been used as a primary in vivo screen for anti-inflammatory activity of most NSAIDs and is believed to be predictive of efficacy in humans. The anti-inflammatory activity of the test pharmaceutical compositions or compounds of the present invention is expressed as percent inhibition of hindpaw weight gain in the test group relative to the vehicle-administered control group.

In a specific embodiment of the invention, the experimental animal model used is an adjuvant-induced arthritis rat model, and the anti-inflammatory activity of the pharmaceutical compositions and compounds of the invention can be determined by measuring body weight in comparison to a control group. Alternatively, the efficacy of the pharmaceutical compositions and compounds of the invention can be assessed by assays that measure bone loss. Animal models such as ovariectomy-induced bone resorption mouse, rat and rabbit models are dynamic parameters known in the art for obtaining bone formation. Bone volume is determined in vivo by methods such as described by Yositake et al or Yamamoto et al using a display microcomputer tomography and bone histomorphometric analysis. Yoshitake et al, "Osteopontin-Deficient MiceAre Resistant to Ovariectomy-Induced Bone Rescription," Proc. Natl. Acad. Sci.96: 8156-; yamamoto et al, "The Integrin Ligand and Echistatin precursors bond Loss in innovative fabricated Mice and rates," Endocrinology139 (3): 1411-1419, (1998), both of which are incorporated herein by reference.

In addition, animal models for inflammatory bowel disease can also be used to assess the efficacy of the pharmaceutical compositions and compounds of the invention (Kim et al, 1992, Scand. J. gastroenterol.27: 529. sub. 537; Strober, 1985, dig. Dis. Sci.30(12 Suppl): 3S-10S). Ulcerative colitis and Crohn's disease are human inflammatory bowel diseases that can be induced in animals. The inflammatory bowel disease can be induced by orally administering sulfated polysaccharides including, but not limited to, pullulan, carrageenan, pullulan sulfate, and dextran sulfate to animals or by chemical stimulants including, but not limited to, trinitrobenzene sulfonic acid (TNBS) and acetic acid.

Animal models for asthma may also be used to assess the efficacy of the pharmaceutical compositions and compounds of the invention. An example of such a model is the murine adoptive transfer model, in which aeroallergen priming of mice receiving either TH1 or TH2 results in migration of TH effector cells to the respiratory tract, with a strong neutrophil (TH1) and eosinophil (TH2) pulmonary mucosal inflammatory response (Cohn et al, 1997, j.exp. med.1861737-1747).

Animal models for psoriasis can also be used to assess the efficacy of the pharmaceutical compositions and compounds of the invention. Animal models of psoriasis have been developed (see, e.g., Schon, 1999, J.Invest.Dermatol.112: 405-410).

In addition, any assay known to those skilled in the art may be used to assess the prophylactic and/or therapeutic use of the pharmaceutical compositions and compounds of the invention for the conditions disclosed herein.

The effect of the pharmaceutical compositions and compounds of the present invention on peripheral blood lymphocyte counts can be monitored/assessed by standard techniques known to those skilled in the art. Peripheral blood lymphocyte counts of an individual can be determined, for example, by obtaining a peripheral blood sample from the individual, separating the lymphocytes from other components of the peripheral blood (e.g., plasma) using, for example, Ficoll-Hypaque (pharmacia) gradient centrifugation, and counting the lymphocytes using the platform blue. Peripheral blood T cells of an individual can be determined as follows: lymphocytes can be separated from other components of peripheral blood (e.g., plasma) by, for example, Ficoll-hypaque (pharmacia) gradient centrifugation, labeled with antibodies (conjugated to FITC or phycoerythrin) against T cell antigens (e.g., CD3, CD4, and CD8), and the number of T cells measured by FACS.

Toxicity and/or efficacy of the pharmaceutical compositions and compounds of the invention can be determined by standard pharmaceutical procedures in cell tissues or experimental animals, e.g., for determining LD₅₀(median lethal dose) and ED₅₀(half therapeutically effective amount). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as LD₅₀/ED₅₀And (4) the ratio. Pharmaceutical compositions and compounds of the invention with high therapeutic indices are preferred. Although the pharmaceutical compositions and compounds of the present invention may be used with toxic side effects, care should be taken to design delivery systems that target such compositions and compounds to the affected tissue sites to reduce potential damage to unaffected cells and thereby reduce side effects.

Data obtained from cell tissue assays and animal studies can be used to formulate dosage ranges for pharmaceutical compositions and compounds of the invention for use in humans. The dosage of such formulations is preferably located to include ED₅₀And a circulating concentration range with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any formulation used in the methods of the invention, the therapeutically effective dose can be estimated initially from a tissue assay. Can be dosed in animal models to obtain IC's determined in cellular tissues ₅₀(concentration of test compound to obtain maximum half-maximal inhibition of symptoms) circulating plasma concentration range. Such information can be used to more accurately determine the dosage for a human. Levels in plasma can be determined by, for example, High Performance Liquid Chromatography (HPLC) and Radioimmunoassay (RIA). The pharmacokinetics of a prophylactic or therapeutic agent can be determined by measuring a parameter such as the peak plasma level (C)_max) Music, yeast, etcArea under the line (AUC, determined by plotting plasma concentration of the formulation against time, reflecting bioavailability), half-life of the compound (t)_1/2) And time of maximum concentration.

Efficacy in preventing or treating a proliferative disorder (e.g., cancer) can be demonstrated, for example, by testing the ability of the pharmaceutical compositions and compounds of the invention to alleviate one or more symptoms of a proliferative disorder, reduce proliferation of cancer cells, slow spread of cancer cells, or reduce tumor size. The efficacy of preventing or treating an inflammatory disorder can be demonstrated, for example, by testing the ability of the pharmaceutical compositions and compounds of the invention to alleviate one or more symptoms of an inflammatory disorder, reduce T cell activation, reduce T cell proliferation, modulate one or more cytokine profiles, reduce cytokine production, reduce inflammation of a joint, organ, or tissue, or improve quality of life. Changes in inflammatory disease activity can be assessed by tender and swollen joint counts, overall patient and attending physician scores for pain and disease activity, and ESR/CRP. The progression of structural joint injury can be assessed by X-ray quantitative scoring (Sharp) of the hands, wrists and feet. Changes in the functional status of persons with inflammatory disorders can be assessed by a Health Assessment Questionnaire (HAQ), while quality of life changes are assessed with SF-36.

4.6 examples

4.6.1 biological assay

The compounds of the invention can be determined by the following examples. Common chemicals as well as the tubulin inhibitors Taxol, vinblastine and colchicine are available from Sigma (st. All compounds were dissolved in 100% DMSO before further dilution by cell culture broth. The final DMSO concentration of all samples, including controls, was kept at a constant 0.1% unless otherwise stated. Streptavidin-coated yttrium SPA beads were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). [³H]Colchicine is obtained from New England Nuclear (Boston, MA), [ 2 ]³H]Paclitaxel and [ alpha ], [ alpha³H]Vinblastine was obtained from Morevek Biochemicals (break, CA).Purified tubulin and biotin-labeled bovine brain tubulin without microtubule-binding protein were obtained from Cytoskeleton, Inc (Denver, CO).

Human tumor cell lines HT29 (colon adenocarcinoma, HTB-38), HT-144 (melanoma, HTB-63), HCT 116 (rectal cancer, CCL-247), a549(NSCLC, CCL 185), NIH: OVCAR-3 (ovarian adenocarcinoma, HTB-161), PC-3 (prostate cancer, CRL-1435), HCT-15 (colorectal adenocarcinoma, CCL-225), MCF-7 (breast cancer, HTB-22), MES-SA (uterine sarcoma, CRL-1976), MES-SA/MX2(CRL-2274), MES-SA/Dx5(CRL-1977) were obtained from American type culture Collection (Manassas, Va.). MCF-7/ADR is supplied by Signal Research division of cell Corporation. All cell lines were at 37 ℃ with 5% CO as published or described in the ATCC information Table ₂The culture medium of (4). Detailed characteristics of human parental MCF-7, MES-SA cell lines and multidrug-resistant, P-gp 170-overexpressing MCF-7/ADR, MES-SA/MX2, MES-SA/Dx5, HCT-15 cell lines have been reported (see Shan, J., Mason, J.M., Yuan, L., Barcia, M., Porti, D., Calabro, A., Budman, D., Vinguiguerra, V., and Xu, H.Rab6c, "A new member of the rab Gene family, is injected in drug resistance in MCF7/AdrR cells", Gene 257: 67-75 (2000)). HUVEC is supplied by Cellular Therapeutic Division of cell Corporation. PBMCs from normal donors were obtained by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density centrifugation.

4.6.1.1 cell proliferation assay

By [ 2 ] in cancer cell lines, HUVEC and human PBMC³H]Thymidine incorporation assay to assess cell proliferation. Briefly, cells were seeded in 96-well microtiter plates 24 hours prior to compound addition to allow them to adhere to the plate. Each compound was tested in triplicate by serial dilution. After compound treatment, the cells were incubated at 37 ℃ for a further 72 hours. Will 2³H]Thymidine (1. mu. Ci in 20. mu.l medium) was added to each well over the last 6 hours of incubation. Cells were subsequently harvested using a TopCount ® Microplate science Coun ter (Packard Instrument Company, Meriden, CT) to detect incorporated tritium. IC was calculated by nonlinear regression analysis using GraphPad Prism ® program (San Diego, Calif.)₅₀。

4.6.1.2 flow cytometry analysis

Cells were harvested after 24 hours treatment with Test formulations and stained with Propidium Iodide (PI) for cell Cycle analysis as described in the Cycle Test Plus DNA Reagentkit of Becton Dickinson (San Jose, CA). Samples were detected using a FACS Calibur instrument (Becton Dickinson, San Jose, Calif.). Cell cycle distribution Using CellQuest^TMv3.1acqusition software and ModFit^TMv2.0program for analysis.

Cells were treated with test formulations for 48 hours, followed by harvesting for apoptosis analysis. FITC-Annexin V binding and DNA were double stained with PI as described previously (see Zhang, L.H.and Long, R.E., "Induction of apoptosis in microorganisms bacteriology A and its synthesis", Life Sci, 64: 1013 1028 (1999)).

4.6.1.3 tubulin polymerization or stability assay

Using CytodYNAMIX^TMScreen (Cytoskeleton, Denver, CO) to monitor the polymerization or stability of purified tubulin. This assay uses 96 well assay plates with 200. mu.g of lyophilized purified tubulin in each well. Tubulin was treated with ice-cold 180. mu.l polymerization or stabilization buffer (80mM PIPES, 1mM MgCl. sub.g.) containing test compound ₂1mM EGTA), or recovered with vehicle control DMSO. The assay was performed in a temperature controlled microtiter plate reader at 37 ℃. Polymerization or stability of tubulin was monitored spectroscopically by the change in absorbance at 340 nm. Using Powerwave^TMThe absorbance was measured by an HT microtiter plate reader (Bio-Tek Instruments, Highland Park, VT) at 1 minute intervals for 60 minutes.

4.6.1.4 immunofluorescence microscopy

Alpha-tubulin in A549 cells was detected by immunofluorescence according to the methods previously described (see Isbrucker, R.A., Gunasekera, S.P., and Longley, R.E, "Structure-activity relationship students of discodermolide and iteratively activated analytes on microorganism function and cytotoxicity", Cancer chemistry. Pharmacol., 48: 29-36 (2001)). Briefly, cells were treated with test compounds for 24 hours and then washed with PBS. The cells were then fixed and permeabilized with warm PBS buffer (3.7% formaldehyde and 1% Triton-X) for 30 minutes. Cells were washed twice with PBS and saturated with 1% mouse blocking serum in PBS for 30 minutes, stained with anti-alpha-tubulin-FITC antibody (Sigma) alone or in the presence of 100 μ g/ml propidium iodide. Cells were visualized under an epifluorescence microscope (Nikon instruments, Melville, NY) and using Image-Pro ^TM(MediaCybernetics, Silver Spring, MD).

4.6.1.5 tubulin competitive binding SPA assay

The use of biotin-labeled tubulin, streptavidin-coated yttrium SPA beads and streptavidin [ supra ] (see Tahir, S.K., Kovar, P., Rosenberg, S.H., and Ng, S.C., "Rapid collagen composition-binding interaction promotion assay, biotin-coated tubulin", Biotechniques, 29: 156-³H]A labeled ligand ([ alpha ])³H]Colchicine [ colchicine ], [ alpha ], [³H]Paclitaxel or [ alpha ], [ alpha ]³H]Vinblastine) for the determination of tubulin binding. Briefly, the binding mixture comprises 0.08 μ M in 100 μ l of assay buffer³H]Labeled ligand, 1mM GTP and 0.5. mu.g biotinylated tubulin, assay buffer containing 80mM PIPES pH 6.9, 1mM MgCl₂1mM EGTA and 5% glycerol. Adding the test compound and the protein prior to tubulin³H]-a labelled ligand. After incubation for 2 hours at 37 ℃, 20 μ l SPA beads (80 μ g in assay buffer) were added. After further incubation with shaking at room temperature for 30 minutes, SPA beads were allowed to settle for 45 minutes in TopCount^®Scintillation counting was performed on a Microplate science Counter.

4.6.1.6caspase enzyme assay

According to the supplier of the detection kit (R)&D systems, Minneapolis, MN) to determine caspase enzyme activity. Briefly, after treatment with the drug, cells were harvested and centrifuged at 250 Xg for 10 minutes. The cell pellet is lysed with lysis buffer. The cell lysate was incubated on ice for 10 minutes, followed by centrifugation at 10,000 Xg for 1 minute. Enzymatic reactions for caspase activity were performed in 96-well microtiter plates. Mu.l of lysate containing 200. mu.g of total protein, 50. mu.l of 2 × reaction buffer and 5. mu.l of caspase-specific peptide substrate (DEVD, IETD or LEHD binding to caspase-3, -8, -9, respectively, p-nitroaniline) were mixed. The mixture was incubated at 37 ℃ for 2 hours, followed by reading A with a microplate reader_405nm. Results are expressed as fold changes in caspase activity between drug-treated cells and vehicle control cells.

4.6.1.7 immunoblot analysis of cell cycle regulatory proteins

Cancer cells were treated with the compounds of the invention or 0.1% DMSO for 24 hours. Cells were trypsinized and spun down in a microcentrifuge for 6 seconds, then immediately lysed in 0.1ml lysis buffer containing 10mM Tris-HCl pH 8.0, 10mM EDTA, 150mM NaCl, 1% NP-40, 0.5% SDS, 1mM DTT, 1mM Na ₃VO₄A cocktail of complete protease inhibitors was added (Roche Applied Science, Indianapolis, IN). Followed by Qiashededer^TM(Qiagen, Valencia, CA) was spun for 1 minute and frozen in dry ice. Samples were diluted with 3 x SDS sample buffer (New England Biolabs, Beverly, MA) and boiled for 5 minutes. About 30. mu.l of this mixture was loaded into each lane of a glycine polyacrylamide gel (Invitrogen, Carlsbad, Calif.), electrophoresed and then transferred onto a PVDF membrane (Invitrogen). PVDF membranes were blocked with PBS containing 0.05% Tween-20 and 5% skim milk powder for 1 hour at room temperature, followed by addition of anti-MPM-2 (Upstate Biotechnology, Lake plain, NY) at 4 deg.C,Bcl-2, Cdc2, p53, p21 or Cdc25C (Santa Cruz Biotechnology, Santa Cruz, Calif.) overnight. The membranes were washed and incubated with HRPO-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, Calif.) (1: 10,00 dilution) for 60 min at room temperature, washed 3 times, and subsequently developed with ECL Plus chemiluminiscence detection system (Amersham biosciences, Piscataway, NJ).

4.6.1.8 ELISA detection of PBMC cultures and TNF-alpha

PBMC were prepared by density centrifugation on Ficoll-Hypaque. PBMC were processed at 1X 10 ⁶Resuspending in complete RPMI-1640 medium/10% fetal calf serum, with or without compound (0.1-100. mu.M) in 24-well plates at 37 ℃ with 5% CO₂The culture was stimulated with LPS (1. mu.g/ml; Escherichia coli serotyp 0127: B8; Sigma) for 24 hours. Cell-free supernatant was collected and aliquoted for storage at-70 ℃ until assayed by ELISA. With R&The assay and reagents provided by DSystems (Minneapolis, MN) measure TNF- α in the supernatant.

4.6.1.9PDE4 detection

PDEs are purified from U937 cells according to the methods described previously (see Marriott, J.B., Westby, M., Cookson, S., Guckian, M., Goodbourn, S., Muller, G., Shire, M.G., Stirling, D., and Dalgleish, A.G., "CC-3052: A water-soluble analog of clinical and potential inhibitor of activation-induced TNF-alpha production", J.Immunol., 161: 4236-. Briefly, cells (1X 10)⁹) Washed with PBS and in cold homogenization buffer (20mM Tris-HCl, pH 7.1, 3mM 2-mercaptoethanol (2-mercaptoranol), 1mM MgCl₂0.1mM EGTA, 1. mu.M PMSF, 1. mu.g/ml leupeptin). After homogenization, the supernatant was collected by centrifugation and loaded onto a Sephacryl S-200 column equilibrated with homogenization buffer. The PDE was eluted in homogenization buffer and the rolipram sensitive segments were pooled and stored in aliquots. Methods described by Di Santo and Heaslip (DiSanto, M.E.and Heaslip, R.J., "Identification and stabilization of large molecular weight PDE-IVs from U937 ", biochem. Biophys. Res. Commun., 197: 1126 + 1131(1993)) and at different concentrations of compound, 50mM Tris-HCl, pH 7.5, 5mM MgCl₂And 1. mu.M cAMP (1% thereof is [, ])³H]cAMP) is determined. The amount of extract used was predetermined to ensure that the reaction was in the linear range and consumed less than 15% of the total substrate. The reaction was carried out at 30 ℃ for 30 minutes and terminated by boiling for 2 minutes. The samples were then quenched and treated with snake venom (1mg/ml) at 30 ℃ for 15 minutes. Unused substrate was removed by addition of 200. mu. lAG1-X8 resin (Bio-Rad, Richmond, Calif.) for 15 min. The sample was then spun at 3000rpm for 5 minutes and 50. mu.l of the aqueous phase was taken for counting. Each data point was double treated and activity was expressed as a percentage of the control. IC (integrated circuit)₅₀Determined by dose-response curves of three independent experiments.

4.6.1.10 human tumor xenograft model

CB 17SCID mice (6-8 weeks old, female) were housed in mini-cages under sterile conditions. Mice were injected subcutaneously with HCT-116 (colon cancer) cells (2X 10) suspended in sterile PBS⁶Cell/mouse). On day 6, tumors of all mice were measured with digital calipers and formula W ²Xl/2 [ W ═ width (minor axis); length (long axis)]Tumor volume was calculated. The combined tumor size is 75-125mm³And randomly distributed into each cage. The mice were then labeled on their ears and each cage was randomly assigned to the treatment group. On day 7, tumors were measured and recorded as initial volumes, after which mice were injected intraperitoneally with vehicle controls (N-methyl-2-pyrrolidone: PEG 400: saline in a ratio of 1: 9: 10), CC-5079(5 and 25mg/kg) or positive controls Camptosar^TM(10 mg/kg). Tumor size was measured at specified time intervals.

4.6.1.11 cell adhesion assay

HUVECs were seeded into 24-well culture plates and cultured for 2 days to form a continuous monolayer of cells. Cancer cells or cancer cell lines (e.g., LS-180 human colon adenocarcinoma cells) were labeled with 5. mu.M calcein-AM for 30 minutes. calcein-AM labeled LS-180 cells were added to each well of HUVEC cultures and incubated at 37 ℃ for 10 minutes. TNF-. alpha. (80ng/ml) was then added and the cultures were incubated for a further 110 min. Non-adherent cells were removed by washing with PBS. The fluorescence intensity of the adhered LS-180 cells in each well was measured by a fluorescent plate reader set for excitation at 485/20nm and emission at 530/25 nm.

4.6.1.12 detection of cell migration and invasion

Cell migration and invasion were determined by a BD BioCoast Angiogenesis System (BD Biosciences, Bedford, MA) based assay. The fluorescence blocking membrane of the insert was a 3 micron pore size PET filter that had been coated with or without BD Matrigel based matrix (for invasion detection). HUVEC (250. mu.l/well) in serum-free medium was added to the upper chamber and the compounds of the invention were added to the bottom well of medium (750. mu.l/well) containing VEGF as a chemoattractant. The cells were then cultured at 37 ℃ for 22 hours. After the end of the culture, the cells were stained with calcein AM for fluorescence measurement.

4.6.1.13 detection of angiogenesis

The effect of the compounds of the invention on angiogenesis was determined by fresh human umbilical cord collected by trained medical personnel. The umbilical cord was sent directly to the laboratory in about 3 hours and the umbilical cord and vessel lumen were flushed with cold basal nutrient medium. The artery is isolated from the umbilical cord in a sterile environment using mechanical methods, forceps, and small surgical scissors. The vessel was cleared of connective tissue and a 1mm length of the vessel ring was cut in the transverse direction. The vascular rings were placed in EGM-2 medium (Clonetics Corp) in 50ml conical-bottomed tubes and sent to Celgene corporation at 4 ℃.6 well tissue culture plates were coated with 250. mu.l matrigel and allowed to stand at 37 ℃ with 5% CO ₂And (5) gelling for 30-45 minutes. The vascular rings were rinsed with EGM-W medium and placed on a Matrigel-coated void, covered with an additional 250. mu.l Matrigel and allowed to gel for 30-45 minutes at 37 ℃. The vessels were cultured in 4ml of EGM-2 for 24 hours to make the tissue suitableNew circumstances are required. After 24 hours of incubation, each vascular ring was treated with 0.1% DMSO as a control, or with different concentrations of the compounds of the invention. The medium was changed twice a week for a total of 3 weeks. The effect of the compounds of the invention was compared to the vascular rings treated with DMSO. The results were analyzed by image-plus software.

4.6.2 Synthesis of exemplary Compounds of the invention

4.6.2.1(E/Z)3- (2, 3-dihydro-benzofuran-5-yl) -3- (3-ethoxy-4-methoxy-phenyl) -acrylonitrile

To a solution of 5-bromo-2, 3-dihydro-benzofuran (1.0g, 5.0mmol) in THF (10mL) at-78 deg.C was added a solution of N-butyllithium in hexane (1.8mL, 2.5N, 4.5mmol) and held for 20 minutes. To the mixture was added a solution of 3-ethoxy-4, N-dimethoxy-N-methyl-benzamide (1.1g, 4.6mmol) in THF (10mL) at-78 deg.C. After 30 minutes, isopropanol (1mL) and water (10mL) were added to the mixture and the cooling bath was removed. The mixture was stirred at room temperature for 20 minutes. The mixture was extracted with ethyl acetate (50mL) and water (50 mL). The aqueous layer was extracted with ethyl acetate (50 mL). The combined organic layers were washed with HCl (1N, 50mL), water (50mL), brine (50mL), MgSO ₄And (5) drying. The solvent was removed to give 2, 3-dihydro-benzofuran-5-yl) - (3-ethoxy-4-methoxy-phenyl) -methanone as an oil (1.23g, 92% yield). The oil was used in the next step without further purification.

To a stirred solution of diethyl cyanomethylphosphonate (1.3mL, 8.2mmol) in THF (15mL) in an ice bath was added dropwise lithium bis (trimethylsilyl) amide (1.0M in THF, 8.3mL, 8.3 mmol). The mixture was stirred at room temperature for 40 minutes. A solution of 2, 3-dihydro-benzofuran-5-yl) - (3-ethoxy-4-methoxy-phenyl) -methanone (1.23g, 4.1mmol) in dry THF (15mL) was added to the mixture. The mixture was refluxed overnight. The solution was poured into ice water (20 mL). The aqueous layer was extracted with ethyl acetate (2X 50 mL). Combination of Chinese herbsThe organic layer was washed with water (50mL), sodium bicarbonate (50mL, saturated), brine (50mL) and dried over magnesium sulfate. The solvent was removed and purified by chromatography (silica gel) to give a solid mixture of (E/Z)3- (2, 3-dihydro-benzofuran-5-yl) -3- (3-ethoxy-4-methoxy-phenyl) -acrylonitrile (1.1g, 83% yield): mp, 49-51 ℃;¹H NMR(DMSO-d₆)δ1.40-1.47(2t，6H，2CH₃)，3.16-3.27(m，4H，2CH₂)，3.89-3.92(2s，6H，2CH₃)，3.98-4.12(2q，4H，2CH₂)，4.58-4.66(m，4H，2CH₂)，5.49-5.50(2s，2H，2CH)，6.73-7.33(m，12H，Ar)；¹³C NMR(DMSO-d₆) δ 14.68, 29.30, 29.39, 55.96, 56.02, 64.50, 71.78, 71.89, 91.11, 91.31, 109.16, 109.27, 110.96, 112.95, 114.18, 122.14, 123.04, 125.45, 126.62, 127.37, 127.72, 129.48, 129.53, 129.87, 130.57, 131.77, 132.22, 147.88, 148.08, 150.77, 151.32, 161.82, 162.51, 162.84, 162.93; analytical calculation C ₂₀H₁₉NO₃: c, 74.75; h, 5.95; n, 4.36. Measured value: c, 74.40; h, 5.95; n, 4.22.

4.6.2.23- (3, 5-dimethoxy-phenyl) -3- (4-methyl-3, 4-dihydro-2H-benzo [1, 4] oxazin-7-yl) -acrylonitrile

To 7-bromo-4-methyl-3, 4-dihydro-2H-benzo [1, 4] benzo at-78 deg.C]To a solution of oxazine (0.9g, 3.9mmol) in THF (10mL) was added N-butyllithium (1.3mL, 2.5N, 3.3 mmol). After 10 min, the solution was added to a solution of 3, 5-dimethoxy-benzoyl chloride (650mg, 3.2mmol) in THF (10mL) at-78 ℃. After 1 hour, the cooling bath was removed and the mixture was allowed to warm to room temperature. To the mixture was added isopropanol (2mL), water (30mL) and ethyl acetate (50 mL). The aqueous layer was extracted with ethyl acetate (50 mL). The combined organic layers were washed with sodium bicarbonate (50mL, saturated), brine (50mL) and dried over magnesium sulfate. The solvent was removed and purified by chromatography (silica gel) to give (3, 5-dimethoxy) as a yellow solid-phenyl) - (4-methyl-3, 4-dihydro-2H-benzo [1, 4]]Oxazin-7-yl) -methanone (320mg, 30% yield):¹HNMR(CDCl₃)δ2.86(s，3H，CH₃)，3.42(t，J＝5Hz，2H，CH₂)，3.82(s，6H，2CH₃)，4.27(t，J＝4Hz，2H，CH₂)，6.60-6.64(m，2H，Ar)，6.85(d，J＝2Hz，2H，Ar)，7.36(d，J＝8Hz，1H，Ar)，7.43(dd，J＝2，9Hz，1H，Ar)。

to a stirred ice bath solution of diethyl cyanomethylphosphonate (0.64mL, 4.0mmol) in THF (10mL) was added dropwise lithium bis (trimethylsilyl) amide (1.0M in THF, 4.1mL, 4.1 mmol). The mixture was stirred at room temperature for 40 minutes. Mixing (3, 5-dimethoxy-phenyl) - (4-methyl-3, 4-dihydro-2H-benzo [1, 4] ]An aqueous solution of oxazin-7-yl) -methanone (640mg, 2.0mmol) in dry THF (8mL) was added to the mixture. The mixture was refluxed overnight. The solution was poured into ice water (20 mL). The aqueous layer was extracted with ethyl acetate (2X 50 mL). The combined organic layers were washed with water (50mL), sodium bicarbonate (50mL, saturated), brine (50mL) and dried over magnesium sulfate. The solvent was removed and purified by chromatography (silica gel) to give (E/Z)3- (3, 5-dimethoxy-phenyl) -3- (4-methyl-3, 4-dihydro-2H-benzo [1, 4] benzo [ E/Z ] a yellow solid]Mixture of oxazin-7-yl) -acrylonitrile (370mg, 55% yield): mp, 119-;¹H NMR(DMSO-d₆)δ2.88(s，3H，CH₃)，2.92(s，3H，CH₃)，3.20-3.36(m，4H，2CH₂)，3.74(s，6H，2CH₃)，4.17-4.25(m，4H，2CH₂)，5.92(s，1H，CH)，6.09(s，1H，CH)，6.41-6.45(m，4H，Ar)，6.60-6.66(m，3H，Ar)，6.72-6.77(m，4H，Ar)，6.83-6.87(m，1H，Ar)；¹³C NMR(DMSO-d₆) δ 37.76, 37.85, 47.97, 55.35, 55.38, 63.99, 64.14, 90.83, 92.74, 100.64, 101.60, 106.98, 107.12, 111.22, 111.27, 114.29, 116.09, 118.82, 122.54, 123.38, 124.85, 125.32, 138.33, 138.93, 139.56, 141.15, 142.74, 143.11, 160.26, 160.28, 161.13, 161.33; analytical calculation C₂₀H₂₀N₂O₃: c, 71.41; h, 5.99; n, 8.33. Measured value: c, 71.37; h, 5.84; and N, 8.30.

4.6.2.3(E/Z)3- (3-ethoxy-4-methoxy-phenyl) -3- (4-methyl-3, 4-dihydro-2H-benzo [1, 4] oxazin-7-yl) -acrylonitrile

To 7-bromo-4-methyl-3, 4-dihydro-2H-benzo [1, 4] benzo at-78 deg.C ]A solution of oxazine (1.8g, 7.9mmol) in THF (15mL) was added a solution of N-butyllithium in hexane (2.6mL, 2.5N, 6.5mmol) and held for 10 minutes. To the mixture was added a solution of 3-ethoxy-4, N-dimethoxy-N-methyl-benzamide (1.4g, 6.0mmol) in THF (10mL) at-78 deg.C. After 30 minutes, isopropanol (2mL) and water (30mL) were added to the mixture and the cooling bath was removed. The mixture was stirred at room temperature for 20 minutes. The mixture was extracted with ethyl acetate (50mL) and water (50 mL). The aqueous layer was extracted with ethyl acetate (50 mL). The combined organic layers were washed with water (50mL), brine (50mL), MgSO₄And (5) drying. Removing solvent, and purifying by chromatography (silica gel) to obtain (3-ethoxy-4-methoxy-phenyl) - (4-methyl-3, 4-dihydro-2H-benzo [1, 4 ]]Oil of oxazin-7-yl) -methanone (1.7g, 87% yield):¹HNMR(CDCl₃)δ1.47(t，J＝7Hz，3H，CH₃)，3.00(s，3H，CH₃)，3.41(t，J＝4Hz，2H，CH₂)，3.94(s，3H，CH₃)，4.15(q，J＝7Hz，2H，CH₂)，4.28(t，J＝5Hz，2H，CH₂)，6.64(d，J＝8Hz，1H，Ar)，6.88(d，J＝8Hz，1H，Ar)，7.31-7.41(m，4H，Ar)。

to a stirred solution of diethyl cyanomethylphosphonate (1.7mL, 10.8mmol) in THF (20mL) in an ice bath was added dropwise lithium bis (trimethylsilyl) amide (1.0M in THF, 11mL, 11 mmol). The mixture was stirred at room temperature for 40 minutes. Reacting (3-ethoxy-4-methoxy-phenyl) - (4-methyl-3, 4-dihydro-2H-benzo [1, 4 ]]A solution of oxazin-7-yl) -methanone (1.7g, 5.2mmol) in anhydrous THF (10mL) was added to the mixture. The mixture was refluxed overnight. The solution was poured into ice water (20 mL). The aqueous layer was extracted with ethyl acetate (2X 50 mL). Are combined with The organic layer was washed with water (50mL), sodium bicarbonate (50mL, saturated), brine (50mL) and dried over magnesium sulfate. Removing the solvent and purifying by chromatography (silica gel) to obtain (E/Z)3- (3-ethoxy-4-methoxy-phenyl) -3- (4-methyl-3, 4-dihydro-2H-benzo [1, 4 ]]Solid mixture of oxazin-7-yl) -acrylonitrile (1.2g, 66% yield): mp, 99-101 ℃;¹H NMR(DMSO-d₆)δ1.31(t，J＝7Hz，6H，2CH₃)，2.88(s，3H，CH₃)，2.92(s，3H，CH₃)，3.30-3.35(m，4H，2CH₂)，3.79(s，3H，CH₃)，3.82(s，3H，CH₃)，3.96-4.04(2q，4H，2CH₂)，4.18-4.26(m，4H，2CH₂)，5.86(s，1H，CH)，5.94(s，1H，CH)，6.63-7.07(m，12H，Ar)；¹³CNMR(DMSO-d₆) δ 14.60, 14.63, 37.79, 37.88, 47.99, 55.48, 55.54, 63.81, 64.02, 64.18, 89.99, 90.67, 111.24, 111.32, 111.48, 112.71, 113.92, 114.76, 116.28, 119.26, 119.33, 122.23, 122.33, 122.74, 123.38, 125.33, 126.29, 129.63, 131.25, 138.17, 138.82, 142.79, 143.11, 147.31, 147.68, 149.91, 150.84, 161.40; analytical calculation C₂₁H₂₂N₂O₃: c, 71.98; h, 6.33; and N, 7.99. Measured value: c, 71.67; h, 6.15; and N, 7.88.

4.6.2.4(E/Z)3- (3-ethoxy-4-methoxy-phenyl) -3- (1-methyl-1H-benzotriazol-5-yl) -acrylonitrile

To a solution of 4-bromo-2-ethoxy-1-methoxy-benzene (1.5g, 6.5mmol) in THF (15mL) at-78 deg.C was added a solution of N-butyllithium in hexane (2.5mL, 2.5N, 6.3mmol) and held for 20 minutes. To the mixture was added a slurry of 1-methyl-1H-benzotriazole-5-carbaldehyde (1.0g, 6.0mmol) in THF (5mL) at-78 deg.C. After 18 hours, isopropanol (2mL) and water (10mL) were added to the mixture and the cooling bath was removed. The mixture was stirred at room temperature for 20 minutes. The mixture was extracted with ethyl acetate (50mL) and water (50 mL). Acetic acid for aqueous layer Extraction with ethyl ester (50 mL). The combined organic layers were washed with water (50mL), brine (50mL), MgSO₄And (5) drying. The solvent was removed to give (3-ethoxy-4-methoxy-phenyl) - (1-methyl-1H-benzotriazol-5-yl) -methanol as an oil (2 g). The oil was used in the next step without further purification.

Mixing the above (3-ethoxy-4-methoxy-phenyl) - (1-methyl-1H-benzotriazol-5-yl) -methanol and MnO₂A mixture (2.5g, 29mmol) in dichloromethane (40mL) was stirred at room temperature for 18 h. Adding more MnO₂(1.5g) and kept overnight. The suspension was filtered through a celite filter. Removal of the solvent gave (3-ethoxy-4-methoxy-phenyl) - (1-methyl-1H-benzotriazol-5-yl) -methanone as an off-white solid (1.12g, 58% yield, 2 steps):¹HNMR(CDCl₃)δ1.49(t，J＝7Hz，3H，CH₃)，3.97(s，3H，CH₃)，4.17(q，J＝7Hz，2H，CH₂)，4.36(s，3H，CH₃)，6.91(d，J＝8Hz，1H，Ar)，7.40(dd，J＝2，8Hz，1H，Ar)，7.50(d，J＝2Hz，1H，Ar)，7.62(d，J＝8Hz，1H，Ar)，8.04(dd，J＝2，9Hz，1H，Ar)，8.44-8.45(m，1H，Ar)。

to a stirred ice bath solution of diethyl cyanomethylphosphonate (1.2mL, 7.6mmol) in THF (12mL) was added dropwise lithium bis (trimethylsilyl) amide (1.0M in THF, 7.6mL, 7.6 mmol). The mixture was stirred at room temperature for 40 minutes. (3-ethoxy-4-methoxy-phenyl) - (1-methyl-1H-benzotriazol-5-yl) -methanone (1.12g, 3.6mmol) was added to the mixture. The mixture was refluxed overnight. The solution was poured into ice water (20 mL). The mixture was stirred with ether (20mL) to give a suspension. Filtration afforded (E/Z) a mixture of 3- (3-ethoxy-4-methoxy-phenyl) -3- (1-methyl-1H-benzotriazol-5-yl) -acrylonitrile as an off white solid (1.16g, 96% yield) (isomer ratio 1: 0.7): mp, 180-; [ minor isomer ] ]¹HNMR(DMSO-d₆)δ1.30(t，J＝7Hz，6H，2CH₃)，3.78(s，3H，CH₃)，[3.84(s，CH₃)]，3.92-4.02(m，2H，CH₂)，[4.32(s，CH₃)]，4.37(s，3H，CH₃)，[6.29(s，CH)]，6.39(s，1H，CH)，6.71(dd，J＝2，8Hz，1H，Ar)，6.92-6.99(m，2.3H，Ar)，7.08-7.11(m，1.6H，Ar)，7.43(dd，J＝2，9Hz，1H，Ar)，[7.51(dd，Ar)]，[7.88(d，Ar)]，7.96(d，J＝9Hz，1H，Ar)，[8.04(s，Ar)]，8.11(s，1H，Ar)；¹³CNMR(DMSO-d₆) δ 14.59, 34.55, 55.56, 63.82, 63.87, 94.63, 95.16, 110.88, 111.04, 111.41, 111.65, 111.93, 113.79, 118.54, 119.51, 119.99, 122.37, 122.47, 127.55, 128.24, 129.30, 130.19, 133.17, 133.73, 134.18, 134.61, 144.88, 145.11, 147.52, 147.95, 150.37, 151.24, 161.03, 151.29; analytical calculation C₁₉H₁₈N₄O₂+0.4H₂O: c, 66.81; h, 5.55; and N, 16.40. Measured value: c, 66.79; h, 5.32; n, 16.28.

4.6.2.5(E/Z)3- (3-ethoxy-4-methoxy-phenyl) -3-quinolin-6-yl-acrylonitrile

To a solution of 4-bromo-2-ethoxy-1-methoxy-benzene (1.74g, 7.5mmol) in THF (15mL) at-78 deg.C was added a solution of N-butyllithium in hexane (3.0mL, 2.5N, 7.5mmol) and held for 20 min. To the mixture was added a solution of quinoline-6-carboxylic acid methoxy-methyl-amide (1.55g, 7.2mmol) in THF (10mL) at-78 deg.C. After 2 hours, isopropanol (2mL) and water (50mL) were added to the mixture and the cooling bath was removed. The mixture was stirred at room temperature for 20 minutes. The solvent was removed and the residue was stirred with ether (20 mL). Filtration gave (3-ethoxy-4-methoxy-phenyl) -quinolin-6-yl-methanone (1.5g) as a yellow solid. The solid was used in the next step without further purification.

To a stirred solution of diethyl cyanomethylphosphonate (1.5mL, 9.5mmol) in THF (15mL) in an ice bath was added dropwise lithium bis (trimethylsilyl) amide (1.0M in THF, 9.5mL, 9.5 mmol). The mixture was stirred at room temperature for 40 minutes. (3-ethoxy-4-methoxy-phenyl) -quinolin-6-yl-methanone (1.46g, 4.8mmol) was added to the mixture.The mixture was refluxed overnight. The solution was poured into ice water (50 mL). The aqueous layer was extracted with ethyl acetate (2X 50 mL). The combined organic layers were washed with water (50mL), sodium bicarbonate (50mL, saturated), brine (50mL) and dried over magnesium sulfate. The solvent was removed and purified by chromatography (silica gel) to give (E/Z) a mixture of 3- (3-ethoxy-4-methoxy-phenyl) -3-quinolin-6-yl-acrylonitrile as an off-white solid (1.25g, 79% yield): mp, 114-;¹HNMR(DMSO-d₆)δ1.30(t，J＝7Hz，6H，2CH₃)，3.79(s，3H，CH₃)，3.85(s，CH₃)，3.93-4.00(2q，4H，2CH₂)，6.33(s，1H，CH)，6.45(s，1H，CH)，6.74(d，J＝8Hz，1H，Ar)，6.95-7.13(m，5H，Ar)，7.54-7.67(m，3H，Ar)，7.78(d，J＝9Hz，1H，Ar)，7.95(s，1H，Ar)，8.03-8.14(m，3H，Ar)，8.40(d，J＝8Hz，1H，Ar)，8.48(d，J＝8Hz，1H，Ar)，8.94-9.00(m，2H，Ar)；¹³C NMR(DMSO-d₆) δ 14.55, 14.58, 55.54, 55.59, 63.82, 63.89, 94.74, 95.84, 111.49, 111.69, 111.93, 113.83, 118.44, 118.47, 122.19, 122.23, 122.45, 122.54, 127.45, 127.54, 128.91, 128.99, 129.12, 129.19, 129.27, 129.93, 130.08, 135.39, 136.38, 136.53, 136.88, 147.56, 147.74, 148.02, 148.20, 150.43, 151.31, 151.67, 151.83, 160.84, 160.96; analytical calculation C ₂₁H₁₈N₂O₂+0.1H₂O: c, 75.93; h, 5.52; n, 8.43. Measured value: c, 75.92; h, 5.48; n, 8.41.

4.6.2.6(E/Z)3- (3, 5-dimethoxy-phenyl) -3-quinolin-6-yl-acrylonitrile

To a solution of 1-bromo-3, 5-dimethoxy-benzene (2.7g, 12mmol) in THF (20mL) at-78 deg.C was added a solution of N-butyllithium in hexane (4.5mL, 2.5N, 11mmol) and held for 20 min. To the mixture was added quinoline-6-carboxylic acid methoxy-methyl-amide (2.2g, 10mmol) at-78 ℃. After 2 hours, water (30mL) was addedThe cooling bath was removed from the mixture. The mixture was stirred at room temperature for 20 minutes. The mixture was extracted with ethyl acetate (2X 50 mL). The combined organic layers were washed with sodium bicarbonate (50mL, saturated), brine (50mL) and dried over magnesium sulfate. The solvent was removed and a slurry was formed in ether to give (3, 5-dimethoxy-phenyl) -quinolin-6-yl-methanone as a white solid (1.48g, 50% crude yield). The sample was used in the next step without purification.¹HNMR(CDCl₃)δ3.84(s，6H，2CH₃)，6.70-6.73(m，1H，Ar)，6.97(d，J＝2Hz，1H，Ar)，7.49(dd，J＝4，8Hz，1H，Ar)，8.17-8.28(m，4H，Ar)，9.02-9.04(m，1H，Ar)。

To a stirred solution of diethyl cyanomethylphosphonate (1.6mL, 10mmol) in THF (10mL) in an ice bath was added dropwise lithium bis (trimethylsilyl) amide (1.0M in THF, 10mL, 10 mmol). The mixture was stirred at room temperature for 40 minutes. A solution of (3, 5-dimethoxy-phenyl) -quinolin-6-yl-methanone (1.48g, 5mmol) in THF (10mL) is added to the mixture. The mixture was refluxed for 2 hours. The solution was poured into ice water (30 mL). The aqueous layer was extracted with ethyl acetate (2X 50 mL). The combined organic layers were washed with water (50mL), sodium bicarbonate (50mL, saturated), brine (50mL) and dried over magnesium sulfate. The solvent was removed and purified by chromatography (silica gel) to give (E/Z) a mixture of 3- (3, 5-dimethoxy-phenyl) -3-quinolin-6-yl-acrylonitrile as an off-white solid (1.46g, 92% yield) (isomer ratio 1: 1 according to HNMR): mp: 142 ℃ and 144 ℃; ¹H NMR(DMSO-d₆)δ3.72(s，6H，2CH₃)，3.77(s，6H，2CH₃)，6.50-6.65(m，6H，Ar，2CH)，6.64-6.70(m，2H，Ar)，7.53-7.69(m，3H，Ar)，7.81-7.85(m，1H，Ar)，7.92(d，J＝1Hz，1H，Ar)，8.03-8.14(m，3H，Ar)，8.38-8.50(m，2H，Ar)，8.93-9.01(m，2H，Ar)；¹³C NMR(DMSO-d₆) δ 97.49, 97.65, 101.33, 102.17, 106.66, 107.27, 117.85, 117.98, 122.26, 127.44, 127.49, 128.35, 128.92, 129.18, 129.26, 129.89, 134.96, 135.44, 136.59, 136.95, 138.79, 139.63, 147.75, 148.21, 151.79, 151.92, 160.53, 160.74, 160.87; analytical calculation C₂₀H₁₆N₂O₂+0.1H₂O: c, 75.50; h, 5.13; and N, 8.80. Measured value: c, 75.33; h, 5.34; n, 8.77.

4.6.2.7(E/Z) -3- (3, 5-dimethoxy-phenyl) -3- (1-methyl-1H-indol-6-yl) -acrylonitrile

To a stirred suspension of 1H-indole-6-carboxylic acid (5.65g, 35.1mmol) in 55mL THF was added 1, 1' -carbonyldiimidazole (6.25g, 38.6 mmol). The suspension was stirred at room temperature for 2 hours, then O, N-dimethyl-hydroxylamine hydrochloride (4.10g, 42.1mmol) was added in the ice bath. The mixture was stirred at room temperature overnight. The mixture was extracted with ethyl acetate (2X 50 mL). The combined ethyl acetate extracts were washed with brine (50mL) and MgSO₄Dried, filtered and concentrated to an oil, which was purified by flash column chromatography (ethyl acetate/hexanes) to give 1H-indole-6-carboxylic acid methoxy-methyl-amide as an oil (5.54g, 77% yield):¹HNMR(CDCl₃)δ3.39(s，3H，NCH₃)，3.58(s，3H，OCH₃) 6.55-6.57(m, 1H, Ar), 7.29-7.84(m, 4H, Ar), 8.85(brs, 1H, NH). The product was used in the next step without purification.

A solution of 1-bromo-3, 5-dimethoxybenzene (12.86g, 59.3mmol) in THF (60mL) was cooled to-78 deg.C, evacuated and refilled with nitrogen 10 times. To the clear solution was added n-butyllithium (23.7mL, 59.3mmol) slowly and stirred for 30 min. A mixture of 1H-indole-6-carboxylic acid methoxy-methyl-amide (5.50g, 26.9mmol) in THF (40mL) was added at-78 deg.C and stirred for 3 hours. The mixture was quenched with isopropanol (12.5mL, 162mmol) and added water (40 mL). Extraction with ether (3 × 50mL), washing with water (2 × 50mL), drying, concentration to an oil, and purification by flash column chromatography (ethyl acetate/hexanes) gave (3, 5-dimethoxy-phenyl) - (1H-indol-6-yl) -methanone as an oil (3.70g, 49% yield). The product was used in the next step without purification.

Oxidizing hydrogen at 0 deg.CPotassium (0.21g, 3.7mmol) was added to a mixture of (3, 5-dimethoxy-phenyl) - (1H-indol-6-yl) -methanone (0.69g, 2.5mmol) in DMF (7mL), followed by methyl iodide (0.2mL, 2.7mmol) and stirring at 0 ℃ for 2H. The mixture was diluted with ether (15mL) and washed with brine (2X 10 mL). The organic phase is MgSO₄Drying, concentration by flash column chromatography (EtOAc/hexanes), purification afforded (3, 5-dimethoxy-phenyl) - (1-methyl-1H-indol-6-yl) -methanone as an off-white solid (0.54g, 75% yield): ¹H NMR(DMSO-d₆)δ3.80(s，6H，2OCH₃)，3.85(s，3H，NCH₃) 6.55(d, J ═ 3Hz, 1H, Ar), 6.78(t, J ═ 2Hz, 1H, Ar), 6.83(d, J ═ 2Hz, 2H, Ar), 7.47(d, J ═ 2Hz, 1H, Ar), 7.61(d, J ═ 3Hz, 1H, Ar), 7.68(d, J ═ 8Hz, 1H, Ar), 7.90(brs, 1H, Ar). The product was used in the next step without purification.

To a solution of diethyl cyanomethylphosphonate (0.55mL, 3.5mmol) in anhydrous THF (8mL) cooled in an ice bath was added lithium bis (trimethylsilyl) amide (1.0M in THF, 3.5mL, 3.5mmol) and stirred at room temperature for 40 minutes, followed by addition of a solution of (3, 5-dimethoxy-phenyl) - (1-methyl-1H-indol-6-yl) -methanone (0.52g, 1.8mmol) in THF (10mL) and reflux overnight. The solution was poured into ice water (10mL) and washed with CH₂Cl₂(2X 50mL), washed with water (30mL), MgSO₄Dried, filtered under vacuum, concentrated to an oil and purified by flash column chromatography (ethyl acetate/hexanes) to give (E/Z) -3- (3, 5-dimethoxy-phenyl) -3- (1-methyl-1H-indol-6-yl) -acrylonitrile as a light yellow solid (0.53g, 94% yield): mp, 117-119 deg.C;¹HNMR(DMSO-d₆)δ3.72-3.80(ms，9H，2OCH₃and NCH₃) 6.27 and 6.32(2s, 1H, CH), 6.46-6.51(m, 3H, Ar), 6.62-6.67(m, 1H, Ar), 6.95-6.99(m, 1H, Ar), 7.47-7.65(m, 3H, Ar); ¹³C NMR(CDCl₃) δ 32.5, 55.3, 93.7, 95.3, 100.6, 101.0, 101.9, 106.8, 107.3, 109.9, 110.8, 118.5, 118.6, 119.4, 120.2, 129.1, 129.6, 129.8, 130.4, 131.8, 132.5, 135.7, 136.1, 139.7, 141.0, 160.3, 163.0. Analytical calculation C₂₀H₁₈N₂O₂: c, 75.45; h, 5.70; and N, 8.80. Measured value: c, 75.31; h, 5.77; and N, 8.66.

4.6.2.8(E/Z) -3- (3-ethoxy-4-methoxy-phenyl) -3- (1H-indol-6-yl) -acrylonitrile

To a stirred brown solution of 1H-indole-6-carboxylic acid (2.68g, 16.6mmol) in THF (25mL) was added CDI (2.97g, 18.3mmol) and stirred at room temperature for 2H. The mixture was cooled to 0 deg.C, O, N-dimethyl-hydroxylamine hydrochloride (1.95g, 20.0mmol) was added, and the mixture was stirred at room temperature overnight. Water (50mL) was added to the reaction and extracted with ethyl acetate (2X 100 mL). The combined organic phases were washed with brine (100mL) and MgSO₄Drying and concentration in vacuo gave 1H-indole-6-carboxylic acid methoxy-methyl-amide as an oil (3.87g, 114% crude product yield):¹H NMR(CDCl₃)δ3.40(s，3H，NCH₃)，3.59(s，3H，OCH₃) 6.57-6.59(m, 1H, Ar), 7.31-7.33(m, 1H, Ar), 7.46-7.50(m, 1H, Ar), 7.64(d, J ═ 8Hz, 1H, Ar), 7.84(s, 1H, Ar), 8.60(brs, 1H, NH). The product was used in the next step without purification.

A stirred mixture of 4-bromo-2-ethoxy-1-methoxy-benzene (6.52g, 23.7mmol) and anhydrous THF (20mL) was cooled to-78 deg.C, evacuated, and refilled with nitrogen 10 times. To the clear solution was added n-butyllithium (9.5mL, 23.7mmol) slowly and stirred for 20 min. A mixture of 1H-indole-6-carboxylic acid methoxy-methyl-amide (2.20g, 10.8mmol) in dry THF (25mL) was added at-78 deg.C and stirred for 1H. The mixture was quenched with isopropanol (4.9mL, 65mmol) and water (15 mL). The mixture was extracted with ethyl acetate (3X 50 mL). The combined organic phases were washed with water (2X 50mL) and MgSO₄Drying and concentration gave an oil which was purified by flash column chromatography (ethyl acetate, hexanes) to give (3-ethoxy-4-methoxy-phenyl) - (1H-indol-6-yl) -methanone as an oil (2.30 g). The product was used in the next step without purificationIn the step (2).

To a solution of diethyl cyanomethylphosphonate (3.6mL, 23mmol) in anhydrous THF (28mL) was added lithium bis (trimethylsilyl) amide (1.0M in THF, 23mL, 23mmol) at 0 deg.C and stirred at room temperature for 30 min, then a solution of (3-ethoxy-4-methoxy-phenyl) - (1H-indol-6-yl) -methanone (2.27g, 70% HPLC purity, 7.68mmol) in THF (15mL) was added and refluxed overnight. The reaction mixture was poured into water (80mL) and CH was used ₂Cl₂(2X 80 mL). The combined organic phases were washed with brine (80mL) and MgSO₄Dried and purified by flash column chromatography (ethyl acetate, hexanes) to give (E/Z) -3- (3-ethoxy-4-methoxy-phenyl) -3- (1H-indol-6-yl) -acrylonitrile as a light yellow solid (0.72g, 30% yield): mp, 132-;¹H NMR(DMSO-d₆)δ1.27-1.33(2t，3H，CH₂CH₃) 3.70(s, 1.9H, OCH of one isomer)₃) 3.79(s, 1.25H, OCH of the other isomer₃)，3.94-4.04(2q，2H，CH₂CH₃) 6.11 and 6.12(2s, 1H, both isomers of CH), 6.48-7.64(m, 8H, Ar), 11.26 and 11.33(2brs, 1H, both isomers of-NH);¹³C NMR(DMSO-d₆) δ 14.58, 55.54, 63.80, 92.17, 92.32, 101.34, 101.39, 111.32, 111.50, 112.54, 112.66, 112.83, 114.02, 118.96, 119.06, 119.15, 119.79, 120.06, 120.30, 122.40, 127.42, 128.04, 128.72, 129.35, 129.75, 130.08, 131.00, 131.40, 135.25, 135.50, 147.36, 147.69, 150.03, 150.90, 163.02, 163.10; analytical calculation C₂₀H₁₈N₂O₂+0.09EtOAc ethyl acetate: c, 74.73; h, 5.87; n, 8.21. Measured value: c, 74.94; h, 5.78; and N, 8.58.

4.6.2.9(E/Z) -3- (3-ethoxy-4-methoxy-phenyl) -3- (1-methyl-1H-indol-6-yl) -acrylonitrile

1H-stirringTo a brown solution of indole-6-carboxylic acid (2.68g, 16.6mmol) in THF (25mL) was added CDI (2.97g, 18.3mmol) and the mixture was stirred at room temperature for 2 hours. The mixture was cooled to 0 deg.C, O, N-dimethyl-hydroxylamine hydrochloride (1.95g, 20.0mmol) was added, and the mixture was stirred at room temperature overnight. Water (50mL) was added to the reaction and extracted with ethyl acetate (2X 100 mL). The combined organic phases were washed with brine (100mL) and MgSO ₄Drying and concentration in vacuo gave 1H-indole-6-carboxylic acid methoxy-methyl-amide as an oil (3.87g, 114% crude product yield):¹H NMR(CDCl₃)δ3.40(s，3H，NCH₃)，3.59(s，3H，OCH₃) 6.57-6.59(m, 1H, Ar), 7.31-7.33(m, 1H, Ar), 7.46-7.50(m, 1H, Ar), 7.64(d, J ═ 8Hz, 1H, Ar), 7.84(s, 1H, Ar), 8.60(brs, 1H, NH). The product was used in the next step without purification.

A stirred mixture of 4-bromo-2-ethoxy-1-methoxy-benzene (6.52g, 23.7mmol) and anhydrous THF (20mL) was cooled to-78 deg.C, evacuated and refilled with nitrogen 10 times. To the clear solution was added n-butyllithium (9.5mL, 23.7mmol) slowly and stirred for 20 min. A mixture of 1H-indole-6-carboxylic acid methoxy-methyl-amide (2.20g, 10.8mmol) in dry THF (25mL) was added at-78 deg.C and stirred for 1H. The mixture was quenched with isopropanol (4.9mL, 65mmol) and water (15 mL). The mixture was extracted with ethyl acetate (3X 50 mL). The combined organic phases were washed with water (2X 50mL) and MgSO₄Drying and concentration gave an oil which was purified by flash column chromatography (ethyl acetate, hexanes) to give (3-ethoxy-4-methoxy-phenyl) - (1H-indol-6-yl) -methanone as an oil (2.30 g). The product was used in the next step without purification.

To a solution of diethyl cyanomethylphosphonate (3.6mL, 23mmol) in anhydrous THF (28mL) was added lithium bis (trimethylsilyl) amide (1.0M in THF, 23mL, 23mmol) at 0 deg.C and stirred at room temperature for 30 min, then a solution of (3-ethoxy-4-methoxy-phenyl) - (1H-indol-6-yl) -methanone (2.27g, 70% HPLC purity, 7.68mmol) in THF (15mL) was added and refluxed overnight. The reaction mixture was poured into water (80mL) and CH was used₂Cl₂(2X 80 mL). The combined organic phases are saltedWashed with water (80mL) and MgSO₄Dried and purified by flash column chromatography (ethyl acetate, hexanes) to give (E/Z) -3- (3-ethoxy-4-methoxy-phenyl) -3- (1H-indol-6-yl) -acrylonitrile as a light yellow solid (0.72g, 30% yield): mp, 132-;¹H NMR(DMSO-d₆)δ1.27-1.33(2t，3H，CH₂CH₃) 3.70(s, 1.9H, OCH of one isomer)₃) 3.79(s, 1.25H, OCH of the other isomer₃)，3.94-4.04(2q，2H，CH₂CH₃) 6.11 and 6.12(2s, 1H, both isomers of CH), 6.48-7.64(m, 8H, Ar), 11.26 and 11.33(2brs, 1H, both isomers of-NH);¹³C NMR(DMSO-d₆) δ 14.58, 55.54, 63.80, 92.17, 92.32, 101.34, 101.39, 111.32, 111.50, 112.54, 112.66, 112.83, 114.02, 118.96, 119.06, 119.15, 119.79, 120.06, 120.30, 122.40, 127.42, 128.04, 128.72, 129.35, 129.75, 130.08, 131.00, 131.40, 135.25, 135.50, 147.36, 147.69, 150.03, 150.90, 163.02, 163.10; analytical calculation C ₂₀H₁₈N₂O₂+0.09 ethyl acetate: c, 74.73; h, 5.87; n, 8.21. Measured value: c, 74.94; h, 5.78; and N, 8.58.

To a solution of (E/Z) -3- (3-ethoxy-4-methoxy-phenyl) -3- (1H-indol-6-yl) -acrylonitrile (0.15g, 0.49mmol) in DMF (2mL) at 0 deg.C was added potassium hydroxide (0.04g, 0.73mmol) and stirred for 8 min, then iodomethane (0.03mL, 0.53mmol) was added. The mixture was stirred at 0 ℃ for 3 h, then diluted with ether (10mL), washed with brine (2X 15mL), and MgSO₄Dried and concentrated to an oil which was purified by flash column chromatography (ethyl acetate/hexanes) to give E/Z-3- (3-ethoxy-4-methoxy-phenyl) -3- (1-methyl-1H-indol-6-yl) -acrylonitrile as a foamy solid (0.15g, 93% yield): mp, 130-;¹H NMR(DMSO-d₆)δ1.27-1.33(2t，3H，CH₂CH₃)，3.78-3.84(ms，6H，OCH₃and NCH₃)，3.93-4.03(2q，2H，CH₂CH₃) 6.14 and 6.19(2s, 1H, CH), 6.46-7.65(m, 8H, Ar);¹³CNMR(DMSO-d₆)δ14.6，14.6，32.5, 55.5, 55.5, 63.7, 63.8, 92.4, 92.8, 100.6, 110.3, 110.9, 111.3, 111.5, 112.4, 114.0, 119.0, 119.1, 119.8, 120.2, 120.4, 122.4, 122.6, 129.0, 129.7, 129.9, 130.0, 131.2, 131.3, 131.6, 132.3, 135.8, 136.1, 147.3, 147.7, 150.2, 151.0, 163.0, 163.2; analytical calculation C₂₁H₂₀N₂O₂: c, 75.88; h, 6.06; n, 8.43. Measured value: c, 75.53; h, 6.09; n, 8.33.

4.6.2.10(E/Z) -3-benzofuran-5-yl-3- (3-ethoxy-4-methoxy-phenyl) -acrylonitrile

A stirred mixture of 4-bromo-2-ethoxy-1-methoxy-benzene (1.74g, 7.5mmol) and dry THF (15mL) was cooled to-78 deg.C, evacuated and refilled with nitrogen 10 times. To the clear solution was added n-butyllithium (3.0mL, 7.5mmol) slowly and stirred for 20 min. A mixture of benzofuran-5-carbaldehyde (1.0g, 6.8mmol) in dry THF (10mL) was added at-78 deg.C and stirred for 1 hour. The mixture was quenched with isopropanol (3.1mL, 41mmol) and added water (10 mL). The mixture was extracted with ethyl acetate (3X 50 mL). The combined organic phases were washed with water (2X 50mL) and MgSO₄Drying and concentration gave benzofuran-5-yl- (3-ethoxy-4-methoxy-phenyl) -methanol as an oil (2.36g, 115% yield). The product was used in the next step without purification.

To a stirred solution of benzofuran-5-yl- (3-ethoxy-4-methoxy-phenyl) -methanol (2.36g crude, 6.8mmol) in CH at room temperature₂Cl₂(15mL) solution activated MnO was added₂Powder (6.0g, 69mmol) and 2-3 equivalents of MnO per 3-5 hours₂Until HPLC showed disappearance of starting material. The black suspension was filtered through a celite pad and concentrated in vacuo to give benzofuran-5-yl- (3-ethoxy-4-methoxy-phenyl) -methanone as an off white solid (2.25g, 111% crude yield). The product was used in the next step without purification.

To a solution of diethyl cyanomethylphosphonate (2.2mL, 13.7mmol) in anhydrous THF (30mL) was added lithium bis (trimethylsilyl) amide (1.0M in THF, 13.7mL, 13.7mmol) at 0 deg.C and stirred at room temperature for 30 minutes, followed by addition of a solution of benzofuran-5-yl- (3-ethoxy-4-methoxy-phenyl) -methanone (2.23g, 6.8mmol) in THF (25mL) and reflux for 2 hours. The reaction mixture was poured into water (20mL) and CH was used₂Cl₂(2X 50 mL). The combined organic phases were washed with brine (50mL) and MgSO₄Dried and purified by flash column chromatography (ethyl acetate/hexanes) to give (E/Z) -3-benzofuran-5-yl-3- (3-ethoxy-4-methoxy-phenyl) -acrylonitrile as a light yellow solid (1.79g, 82% yield): mp, 95-97 ℃;¹H NMR(DMSO-d₆)δ1.31(t，J＝7Hz，3H，CH₂CH₃) 3.79 and 3.84(2s, 3H, OCH)₃)，3.93-4.04(2q，2H，CH₂CH₃) 6.15 and 6.28(2s, 1H, CH), 6.71-8.11(m, 8H, Ar);¹³C NMR(DMSO-d₆) δ 14.5, 14.6, 55.5, 55.6, 63.8, 93.7, 93.9, 107.0, 107.1, 111.4, 111.4, 111.6, 112.0, 113.8, 118.7, 118.7, 122.0, 122.4, 122.5, 125.0, 125.6, 127.4, 127.4, 129.7, 130.7, 132.2, 133.6, 147.1, 147.3, 147.4, 147.8, 150.3, 151.1, 154.6, 155.3, 161.8, 162.1. Analytical calculation C₂₀H₁₇NO₃: c, 75.22; h, 5.37; n, 4.39. Measured value: c, 75.20; h, 5.30; n, 4.41.

4.6.2.113- (3, 5-dimethoxy-phenyl) -3- (1, 2-dimethyl-1H-benzimidazol-5-yl) -acrylonitrile (E and Z isomers)

Grignard reagents were prepared in an oven-dried three-necked flask equipped with a reflux condenser, dropping funnel and magnetic stirrer. A solution of 3, 5-dimethoxy-bromobenzene (2.0g, 9.0mmol) in THF (10mL) was added to a mixture of magnesium strip (0.2g, 9.0mmol) in THF (5mL) and a small piece of iodineIn the composition. The resulting mixture was refluxed for about 3 hours and then cooled to room temperature for about 30 minutes. (3, 5-Dimethoxyphenyl) magnesium bromide was then added slowly to a stirred solution of 1, 2-dimethoxy-1H-benzimidazole-5-carbaldehyde (1.3g, 7.5mmol) in THF (10mL) at 0 deg.C. After the addition was complete, the solution was stirred at room temperature for about 1 hour. The mixture was cooled to 0 ℃ and saturated NH₄Cl solution (40mL) was quenched. The aqueous layer was extracted with ethyl acetate (3X 20 mL). The combined organic layers were washed with water (2X 30mL), brine (30mL) and dried (MgSO₄). The solvent was removed and the crude product was slurried in hexane to give (3, 5-dimethoxy-phenyl) - (1, 2-dimethyl-1H-benzimidazol-5-yl) -methanol (2.1g, 91%) as an off-white solid:¹H NMR(CDCl₃)δ7.66(s，1H)，7.24-7.20(dd，J＝1，8Hz，1H)，7.17(d，J＝8Hz，1H)，6.56(d，J＝2Hz，2H)，6.31(t，J＝2Hz，1H)，5.84(s，1H)，3.72(s，6H)，3.63(s，3H)，3.55(b，1H)，2.52(s，3H)。

(3, 5-dimethoxy-phenyl) - (1, 2-dimethyl-1H-benzimidazol-5-yl) -methanol (2.1g, 6.7mmol) and MnO ₂(2.9g, 33.6mmol) of CH₂Cl₂The suspension (300mL) was stirred at room temperature for 17 h. The mixture was filtered through celite and the solvent was removed. The crude product was slurried with diethyl ether to give (3, 5-dimethoxy-phenyl) - (1, 2-dimethyl-1H-benzoimidazol-5-yl) -methanone (2.0g, 99%) as an off-white solid:¹H NMR(DMSO-d₆)δ7.90(s，1H)，7.71-7.62(m，2H)，6.80(s，3H)，3.80(s，3H)，3.35(s，3H)，2.58(s，3H)；¹³C NMR(DMSO-d₆)δ195.28，160.17，154.83，141.54，140.27，139.15，130.15，123.50，120.73，109.70，107.19，103.62，55.47，29.96，13.52。

3- (3, 5-dimethoxy-phenyl) -3- (1, 2-dimethyl-1H-benzimidazol-5-yl) -acrylonitrile (E and Z isomers) Using a procedure analogous to that for the preparation of 3- (3-amino-4-methoxy-phenyl) -3- (3, 4-dimethoxy-phenyl) -acrylonitrile (E and Z isomers) with (3, 5-dimethoxy-phenyl) - (1, 2-dimethyl-1H-benzimidazol-5-yl) -methanone (2.0g, 6.4mmol), bis (trimethylsilane)Yl) lithium amide (7.7mL, 7.7mmol) and diethyl cyanomethylphosphonate (1.4g, 7.7 mmol). Flash column chromatography of the crude product (silica gel, CH)₂Cl₂∶CH₃OH 95: 5) to yield a white solid as an isomeric mixture of 3- (3, 5-dimethoxy-phenyl) -3- (1, 2-dimethyl-1H-benzimidazol-5-yl) -acrylonitrile (1.1g, 50%): mp 199-;¹H NMR(CDCl₃)δ7.69(m，3H)，6.56-6.42(m，3H)，5.70(5.74)(s，1H)，3.77(s，3H)，3.73(3.74)(s，6H)，2.61(s，3H)；¹³C NMR(CDCl₃) δ 163.76, 160.63(160.60), 153.22(153.54), 142.35(142.57), 141.69(139.43), 137.04(137.38), 130.69(132.54), 123.74(122.64), 120.81(119.54), 118.23(118.09), 108.84(108.80), 107.02(107.72), 102.08(102.02), 94.32(93.47), 55.42(55.41), 30.05(30.00), 13.88; analytical calculation C ₂₀H₁₉N₃O₂+0.2H₂O: c, 71.28; h, 5.80; n, 12.47. Measured value: c, 71.18; h, 5.86; n, 12.42.

The equivalence statement:

the invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art. Such modifications are intended to fall within the scope of the appended claims.

Each of the publications cited herein, the disclosure of which is incorporated herein by reference.

Claims (30)

1. A compound having the formula:

wherein:

x is a substituted or unsubstituted imidazole, a substituted or unsubstituted pyridine, a substituted or unsubstituted pyrrolidine, a substituted or unsubstituted thiophene, a substituted or unsubstituted indole, a substituted or unsubstituted 2, 3-dihydrobenzofuran, a substituted or unsubstituted 3, 4-dihydro-2H-benzo (b) (1, 4) oxazine, a substituted or unsubstituted 1H-benzo (d) (1, 2, 3) triazole, a substituted or unsubstituted quinoline, a substituted or unsubstituted benzofuran, a substituted or unsubstituted benzo (d) oxazol-2 (3H) one, or a substituted or unsubstituted pyrimidine;

R₁and R₂Each independently is-H, -CN, halogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -NHC (O) R₉、-NHC(O)OR₉-COOH, -C (O) -lower alkyl, -C (O) O-lower alkyl, -C (O) -N (R)₉)₂Substituted or unsubstituted aryl, or substituted or unsubstituted heterocycle;

R_aand R_bEach independently is-H, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, halogen, cyano, -NO ₂、-OH、-OPO(OH)₂、-N(R₉)₂、-OC(O)-R₁₀、-OC(O)-R₁₀-N(R₁₀)₂、-C(O)N(R₁₀)₂、-NHC(O)-R₁₀、-NHS(O)₂-R₁₀、-S(O)₂-R₁₀、-S(O)₂-NH₂、-S(O)₂-N(R₁₀)₂、-NHC(O)NH-R₁₀、-NHC(O)N(R₁₀)₂、-NHC(O)NHSO₂-R₁₀、-NHC(O)-R₁₀-N(R₁₀)₂、-NHC(O)CH(R₁₀)(N(R₉)₂) or-NHC (O) -R₁₀-NH₂；

R₃is-H, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, halogen, cyano, -NO₂、-OH、-OPO(OH)₂、-N(R₉)₂、-OC(O)-R₁₀、-OC(O)-R₁₀-N(R₁₀)₂、-OC(O)-R₁₀-NH₂、-C(O)N(R₁₀)₂、-NHC(O)-R₁₀、-NHS(O)₂-R₁₀、-S(O)₂-R₁₀、-OS(O)₂-R₁₀、-S(O)₂-NH₂、-S(O)₂-N(R₁₀)₂、-OS(O)₂-NH₂、-OS(O)₂-N(R₁₀)₂、-NHC(O)O-R₁₀、-NHC(O)NH-R₁₀、-NHC(O)N(R₁₀)₂、-NHC(O)NHSO₂-R₁₀、-NHC(O)-R₁₀-N(R₁₀)₂、-NHC(O)CH(R₁₀)(N(R₉)₂) or-NHC (O) -R₁₀-NH₂Or R is₃And R_aOr R₄Together form-O-C (R)₁₆R₁₇)O-、-O-(C(R₁₆R₁₇))₂-O-or-O- (C (R)₁₆R₁₇))₃-O-；

R₄is-H, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, halogen, cyano, -NO₂、-OH、-OPO(OH)₂、-N(R₉)₂、-OC(O)-R₁₀、-OC(O)-R₁₀-N(R₁₀)₂、-OC(O)-R₁₀-NH₂、-C(O)N(R₁₀)₂、-NHC(O)-R₁₀、-NHS(O)₂-R₁₀、-S(O)₂-R₁₀、-OS(O)₂-R₁₀、-S(O)₂-NH₂、-S(O)₂-N(R₁₀)₂、-OS(O)₂-NH₂、-OS(O)₂-N(R₁₀)₂、-NHC(O)O-R₁₀、-NHC(O)NH-R₁₀、-NHC(O)N(R₁₀)₂、-NHC(O)NHSO₂-R₁₀、-NHC(O)-R₁₀-N(R₁₀)₂、-NHC(O)CH(R₁₀)(N(R₉)₂) or-NHC (O) -R₁₀-NH₂；

R₅is-H, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, halogen,Cyano, -NO₂、-OH、-OPO(OH)₂、-N(R₉)₂、-OC(O)-R₁₀、-OC(O)-R₁₀-N(R₁₀)₂、-OC(O)-R₁₀-NH₂、-C(O)N(R₁₀)₂、-NHC(O)-R₁₀、-NHS(O)₂-R₁₀、-S(O)₂-R₁₀、-OS(O)₂-R₁₀、-S(O)₂-NH₂、-S(O)₂-N(R₁₀)₂、-OS(O)₂-NH₂、-OS(O)₂-N(R₁₀)₂、-NHC(O)O-R₁₀、-NHC(O)NH-R₁₀、-NHC(O)N(R₁₀)₂、-NHC(O)NHSO₂-R₁₀、-NHC(O)-R₁₀-N(R₁₀)₂、-NHC(O)CH(R₁₀)(N(R₉)₂) or-NHC (O) -R₁₀-NH₂；

Each R₉independently-H, substituted or unsubstituted lower alkyl, or substituted or unsubstituted cycloalkyl;

each R₁₀Independently is substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted lower hydroxyalkyl, or R₁₀And the nitrogen atom to which it is attached form a substituted or unsubstituted heterocyclic ring, or R as appropriate ₁₀is-H;

R₁₆and R₁₇Each independently is-H or halogen; and

wherein when:

(1) x is pyridine, substituted pyridine, pyrrolidine, imidazole, naphthalene or thiophene;

(2)R_aand R_bIs H; and

(3)R₄when hydrogen, nitro, cyano, trifluoromethyl, ethoxycarbonyl, methoxycarbonyl, propoxycarbonyl, acetyl, carbamoyl, acetoxy, carboxyl, hydroxyl, amino, lower alkyl, lower alkylenemethyl, lower alkoxy, or halogen,

if R is₃Or R₅One is H, then the other is not-O-C_1-10Alkyl, -O-C_1-10Monocyclic alkyl、-O-C_1-10Polycyclic alkyl radicals, -O-C_1-10Benzocycloalkyl, -C_0-3-C_1-10Alkyl, -C_0-3-C_1-10Monocycloalkyl, -C_0-3-C_1-10Polycyclic alkyl radical, -C_0-3-C_1-10Benzocycloalkyl, -CH ═ C_1-10Alkyl, -CH ═ C_1-10Monocyclic alkyl or-CH ═ C_1-10A bicycloalkyl group.

2. A compound having the formula:

wherein:

x is a substituted or unsubstituted indole, a substituted or unsubstituted 2, 3-dihydrobenzofuran, a substituted or unsubstituted 3, 4-dihydro-2H-benzo (b) (1, 4) oxazine, a substituted or unsubstituted 1H-benzo (d) (1, 2, 3) triazole, a substituted or unsubstituted quinoline, a substituted or unsubstituted benzofuran, a substituted or unsubstituted benzo (d) oxazol-2 (3H) one, or a substituted or unsubstituted pyrimidine;

R₁And R₂Each independently is-H, -CN, halogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, -NHC (O) R₉、-NHC(O)OR₉-COOH, -C (O) -lower alkyl, -C (O) O-lower alkyl, -C (O) -N (R)₉)₂Substituted or unsubstituted aryl, or substituted or unsubstituted heterocycle;

R_aand R_bEach independently is-H, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, halogen, cyano, -NO₂、-OH、-OPO(OH)₂、-N(R₉)₂、-OC(O)-R₁₀、-OC(O)-R₁₀-N(R₁₀)₂、-C(O)N(R₁₀)₂、-NHC(O)-R₁₀、-NHS(O)₂-R₁₀、-S(O)₂-R₁₀、-S(O)₂-NH₂、-S(O)₂-N(R₁₀)₂、-NHC(O)NH-R₁₀、-NHC(O)N(R₁₀)₂、-NHC(O)NHSO₂-R₁₀、-NHC(O)-R₁₀-N(R₁₀)₂、-NHC(O)CH(R₁₀)(N(R₉)₂) or-NHC (O) -R₁₀-NH₂；

R₃is-H, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, halogen, cyano, -NO₂、-OH、-OPO(OH)₂、-N(R₉)₂、-OC(O)-R₁₀、-OC(O)-R₁₀-N(R₁₀)₂、-OC(O)-R₁₀-NH₂、-C(O)N(R₁₀)₂、-NHC(O)-R₁₀、-NHS(O)₂-R₁₀、-S(O)₂-R₁₀、-OS(O)₂-R₁₀、-S(O)₂-NH₂、-S(O)₂-N(R₁₀)₂、-OS(O)₂-NH₂、-OS(O)₂-N(R₁₀)₂、-NHC(O)O-R₁₀、-NHC(O)NH-R₁₀、-NHC(O)N(R₁₀)₂、-NHC(O)NHSO₂-R₁₀、-NHC(O)-R₁₀-N(R₁₀)₂、-NHC(O)CH(R₁₀)(N(R₉)₂) or-NHC (O) -R₁₀-NH₂Or R is₃And R_aOr R₄Together form-O-C (R)₁₆R₁₇)O-、-O-(C(R₁₆R₁₇))₂-O-or-O- (C (R)₁₆R₁₇))₃-O-；

R₄is-H, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, halogen, cyano, -NO₂、-OH、-OPO(OH)₂、-N(R₉)₂、-OC(O)-R₁₀、-OC(O)-R₁₀-N(R₁₀)₂、-OC(O)-R₁₀-NH₂、-C(O)N(R₁₀)₂、-NHC(O)-R₁₀、-NHS(O)₂-R₁₀、-S(O)₂-R₁₀、-OS(O)₂-R₁₀、-S(O)₂-NH₂、-S(O)₂-N(R₁₀)₂、-OS(O)₂-NH₂、-OS(O)₂-N(R₁₀)₂、-NHC(O)O-R₁₀、-NHC(O)NH-R₁₀、-NHC(O)N(R₁₀)₂、-NHC(O)NHSO₂-R₁₀、-NHC(O)-R₁₀-N(R₁₀)₂、-NHC(O)CH(R₁₀)(N(R₉)₂) or-NHC (O) -R₁₀-NH₂；

R₅is-H, substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, halogen, cyano, -NO ₂、-OH、-OPO(OH)₂、-N(R₉)₂、-OC(O)-R₁₀、-OC(O)-R₁₀-N(R₁₀)₂、-OC(O)-R₁₀-NH₂、-C(O)N(R₁₀)₂、-NHC(O)-R₁₀、-NHS(O)₂-R₁₀、-S(O)₂-R₁₀、-OS(O)₂-R₁₀、-S(O)₂-NH₂、-S(O)₂-N(R₁₀)₂、-OS(O)₂-NH₂、-OS(O)₂-N(R₁₀)₂、-NHC(O)O-R₁₀、-NHC(O)NH-R₁₀、-NHC(O)N(R₁₀)₂、-NHC(O)NHSO₂-R₁₀、-NHC(O)-R₁₀-N(R₁₀)₂、-NHC(O)CH(R₁₀)(N(R₉)₂) or-NHC (O) -R₁₀-NH₂；

Each R₉independently-H, substituted or unsubstituted lower alkyl, or substituted or unsubstituted cycloalkyl;

each R₁₀Independently is substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted lower hydroxyalkyl, or R₁₀And the nitrogen atom to which it is attached form a substituted or unsubstituted heterocyclic ring, or R is, where appropriate₁₀is-H; and

R₁₆and R₁₇Each independently is-H or halogen.

3. A compound having the structure:

4. the compound of claim 1, 2 or 3, wherein the compound is the E isomer.

5. The compound of claim 1, 2 or 3, wherein the compound is the Z isomer.

6. A pharmaceutical composition comprising a compound of claim 1 or 2 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the composition is suitable for parenteral, transdermal, mucosal, nasal, buccal, rectal, sublingual, or oral administration to a subject.

8. A method of inhibiting angiogenesis, said method comprising administering to a subject in need thereof an effective amount of a compound of claim 1 or 2.

9. A method of inhibiting or reducing tubulin polymerization or tubulin stability in a cell, comprising contacting the cell with the compound of claim 1 or 2.

10. The method of claim 9, wherein the cell is a cancer cell.

11. A method of inhibiting PDE4 activity in a cell, the method comprising contacting the cell with a compound of claim 1 or 2.

12. A method of inhibiting or reducing tubulin polymerization or tubulin stability in a cell and inhibiting PDE4 activity in a cell, the method comprising contacting the cell with the compound of claim 1 or 2.

13. A method of inhibiting or reducing tubulin polymerization or tubulin stability in a cell and inhibiting tumor necrosis factor- α (TNF- α) activity in a cell, the method comprising contacting the cell with the compound of claim 1 or 2.

14. A method of treating or ameliorating an inflammatory disorder, the method comprising administering to a subject in need thereof an effective amount of a compound of claim 1 or 2.

15. The method of claim 14, wherein the inflammatory disorder is asthma, an allergic disorder, an inflammatory disorder characterized by type 1 mediated inflammation, an inflammatory disorder characterized by type 2 mediated inflammation, a fibrotic disease, pulmonary fibrosis, psoriasis, multiple sclerosis, systemic lupus erythematosus, Chronic Obstructive Pulmonary Disease (COPD), encephalitis, inflammatory bowel disease, crohn's disease, ulcerative colitis disease, ischemic reperfusion injury, gout, behcet's disease, septic shock, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, rheumatoid arthritis (juvenile and adult), osteoarthritis, psoriatic arthritis, inflammatory osteolysis, sepsis, meningitis, and chronic inflammation resulting from chronic viral or bacterial infection.

16. A method of treating or ameliorating cancer, the method comprising administering to a subject in need thereof an effective amount of a compound of claim 1 or 2.

17. A method of inhibiting the proliferation of a cancer cell, the method comprising contacting a cancer cell with an effective amount of a compound of claim 1 or 2.

18. A method of inhibiting the proliferation of multi-drug resistant cancer cells, the method comprising contacting multi-drug resistant cancer cells with an effective amount of a compound of claim 1 or 2.

19. A method of targeting, blocking or disrupting the function of tumor vasculature, the method comprising contacting a tumor with an effective amount of a compound of claim 1 or 2.

20. A method of targeting, blocking or destroying the vascular endothelium of a tumor, said method comprising contacting the tumor with an effective amount of a compound of claim 1 or 2.

21. A method of targeting, blocking or disrupting the function of tumor vasculature and inhibiting angiogenesis in a tumor, comprising contacting the tumor with an effective amount of a compound of claim 1 or 2.

22. A method of treating or ameliorating a central nervous system disorder, the method comprising administering to a subject in need thereof an effective amount of a compound of claim 1 or 2.

23. The method of claim 22, wherein the central nervous system disorder is parkinson's disease, bradykinesia, myotonia, parkinsonian tremor, parkinsonian gait disorder, motor freezing (movement freezing), depression, long-term memory loss, rubinstein-taber syndrome (RTS), dementia, sleep disorders, postural instability, hypokinesia, inflammation, synuclein abnormalities, multiple system atrophy, striatal substantia nigra degeneration, olivopontocerebellar atrophy, Shy-Drager syndrome, motor neuron disease with parkinsonism characteristics, lewy body dementia, Tau pathological disorder, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, amyloidosis disorder, mild cognitive dysfunction, alzheimer's disease with parkinsonism, Genetic disorders which may be characteristic of Parkinson's disease, Wilson's disease, Hallervorden-Spatz disease, Chediak-Hagashi disease, SCA-3 spinocerebellar ataxia, X-linked dystonia Parkinson's syndrome, Huntington's disease, prion diseases, hyperkinesias, chorea, ballism, dystonic tremor, Amyotrophic Lateral Sclerosis (ALS), central nervous system trauma, or myoclonus.

24. A method of treating or ameliorating a refractory cancer, the method comprising administering to an individual in need thereof an effective amount of a compound of claim 1 or 2.

25. The method of claim 24, wherein the compound is the E isomer.

26. The method of claim 24, wherein the compound is the Z isomer.

27. The method of claim 24, wherein the cancer is refractory to colchicine, taxanes (taxanes), or vinblastine.

28. The method of claim 16 or 24, further comprising administering to the individual an effective amount of one or more other anti-cancer agents.

29. The method of claim 28, wherein the at least one anti-cancer agent is paclitaxel, docetaxel (taxotere), cisplatin, carboplatin, oxaliplatin, doxorubicin, gemcitabine, capecitabine, 5-fluorouracil, etoposide, cyclophosphamide, vincristine, vinblastine, topotecan, or irinotecan.

30. The method of claim 28, wherein the at least one anti-cancer agent is an anti-angiogenic agent, a vascular targeting agent, an immunomodulatory agent, or an anti-inflammatory agent.