WO2016023508A1

WO2016023508A1 - Vascular disruption angent with low cytotoxic activity

Info

Publication number: WO2016023508A1
Application number: PCT/CN2015/086871
Authority: WO
Inventors: Chien-Chih OU; Muh-Hwan Su; Chi-Ling LIU
Original assignee: Syncore Biotechnology Co., Ltd; Cancap Pharmaceutical Ltd.
Priority date: 2014-08-13
Filing date: 2015-08-13
Publication date: 2016-02-18
Also published as: CN106660952A; TW201609643A; TWI594982B

Abstract

The present application discloses a compound of formula (I), a polymorph, a tautomer, an enantiomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof for blocking blood stream: wherein each of R₁-R₆, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(O) R, OC (O) OR, OC (O) NRR', SO₂R, SO₃R, SO₂NRR', SR, NRR', NRSO₂NR'R", NRSO₂R', NRSO₃R', NRC (O) R', NRC (O) NR'R", NRC (O) OR', NRC (N) NR'R", C (O) R, C (O) OR, C (O) NRR'; and wherein each of R, R', and R", independently, is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, cyclyl, or heterocyclyl.

Description

VASCULAR DISRUPTION ANGENT WITH LOW CYTOTOXIC ACTIVITY

TECHNICAL FIELD OF THE INVENTION [0001] The present invention relates to use of a chemical compound for treatment as a vascular disruption agent.

BACKGROUND [0002] The functional vascular networks, which offer the necessary nutrition and oxygen to sustain the homeostasis in the whole body, play crucial role for all tissues development, growth, survival, expansion and repair. Furthermore, the abnormal composition, distribution, and assembly of vasculatures have also been found to be associated with several diseases, such as cancers, metabolic diseases, and immunological diseases, etc. Therefore, targeting vasculatures represents a new frontier useful in some clinical applications through the development of molecular and biomedical techniques and therapeutic medicines. [0003] Vasculatures-targeting agents can be categorized into vascular targeting agents (VTAs) (e.g., anti-angiogenesis agents) and vascular disrupting agents (VDAs) (Dumontet and Jordan, 2010; Hollebecque et al., 2012; Lorusso et al., 2011). There are some currently well-characterized useful VTAs and VDAs in oncology represented by anti-mitotic agents, such as taxanes (e.g., paclitaxel and docetaxel) and Combretastatin A4 (CA4). However, these agents currently under development exhibit a narrow therapeutic window with the issues of high toxicity, complex structures and syntheses, drug resistance, isolation procedures, and side effects for clinical treatment. For example, Colchicine, one of potent class of cancer therapeutic drug, has not shown intrinsic value as a clinically applicable anticancer therapeutic due to a high level of toxicity and consequent very narrow therapeutic index. Therefore, discovery of new helpful and useful compounds, which show a wider therapeutic window, may lead to attainment of improving the issues mentioned above. SUMMARY [0004] According to one aspect of the invention, it relates to a compound of formula (I), a polymorph, a tautomer, an enantiomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof for blocking blood stream:

[0005] wherein each of R1– R6, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(O)R, OC(O)OR, OC(O)NRR', SO2R, SO3R, SO2NRR', SR, NRR', NRSO2NR'R", NRSO₂R', NRSO₃R', NRC(O)R', NRC(O)NR'R", NRC(O)OR', NRC(N)NR'R", C(O)R, C(O)OR, C(O)NRR'; and [0006] wherein each of R, R', and R", independently, is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, cyclyl, or heterocyclyl. [0007] The compound of the above, wherein each of R₁- R₆, independently, is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, heteroaryl, cyclyl, or heterocyclyl. [0008] The one or more compound of the above, wherein R is hydrogen, halogen, or methyl. [0009] The one or more compound of the above, wherein R1-R6, independently, is hydrogen or halogen. [0010] The one or more compound of the above, wherein the compound blocks blood stream to cells of non-cancer tissue. A disorder or disease which benefits from blocking blood stream to cells of non-cancer tissue may include aneurysm, arteriovenous malformation, venous malformation, lymphatic malformation, hemangioma, ischemic retinopathy, diabetic retinopathy, choroidal neovascularization, wet age-related macular degeneration and other related disorders. [0011] The one or more compound of the above, wherein the compound blocks blood stream to cells of cancerous tissue. A disorder or disease which benefits from blocking blood stream to cells of cancerous tissue may include bone cancer, brain and CNS tumor, breast cancer, breast cancer, colorectal cancer, endocrine cancer, gastrointestinal cancer, genitourinary cancer, gynaecological cancer, head and neck cancer, leukaemia, lung cancer, lymphoma, eye cancer, skin cancer, soft tissue sarcoma, urinary system cancer, and other types or related disorders. [0012] According to another aspect of the invention, it relates to a pharmaceutical composition for blocking blood stream, comprising a therapeutically effective amount of the compound of any of the above and a pharmaceutical acceptable carrier. [0013] The pharmaceutical composition of the above, wherein a therapeutically effective amount of the compound of any of the above is between 5 mg/kg and 50 mg/kg. [0014] According to another aspect of the invention, it relates to use of a compound of any of the above or a pharmaceutical composition of the above in manufacturing a medicament for blocking blood stream. [0015] According to another aspect of the invention, it relates to a method of blocking blood stream, comprising: contacting a cell with a compound of any of the above or a pharmaceutical composition of the above. [0016] According to another aspect of the invention, it relates to a compound of formula (I), a polymorph, a tautomer, an enantiomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof for inhibiting polymerization of Į -tubulin:

(I) [0017] wherein each of R₁– R₆, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(O)R, OC(O)OR, OC(O)NRR', SO2R, SO3R, SO2NRR', SR, NRR', NRSO2NR'R", NRSO2R', NRSO3R', NRC(O)R', NRC(O)NR'R", NRC(O)OR', NRC(N)NR'R", C(O)R, C(O)OR, C(O)NRR'; and [0018] wherein each of R, R', and R", independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, cyclyl, or heterocyclyl. [0019] According to another aspect of the invention, it relates to a pharmaceutical composition for inhibiting polymerization of Į -tubulin, comprising a therapeutically effective amount of the compound of any of the above and a pharmaceutical acceptable carrier. [0020] According to another aspect of the invention, it relates to use of a compound of any of the above or a pharmaceutical composition of the above in manufacturing a medicament for inhibiting polymerization of Į -tubulin. [0021] According to another aspect of the invention, it relates to a method of inhibiting polymerization of Į -tubulin, comprising: contacting a cell with a compound of any of the above or a pharmaceutical composition of the above. [0022] According to another aspect of the invention, it relates to a compound of formula (I), a polymorph, a tautomer, an enantiomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof for inhibiting capillary-like tube formation:

(I) [0023] wherein each of R1– R6, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(O)R, OC(O)OR, OC(O)NRR', SO2R, SO3R, SO2NRR', SR, NRR', NRSO2NR'R", NRSO₂R', NRSO₃R', NRC(O)R', NRC(O)NR'R", NRC(O)OR', NRC(N)NR'R", C(O)R, C(O)OR, C(O)NRR'; and [0024] wherein each of R, R', and R", independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, cyclyl, or heterocyclyl. [0025] According to another aspect of the invention, it relates to a pharmaceutical composition for inhibiting capillary-like tube formation, comprising a therapeutically effective amount of the compound of any of the above and a pharmaceutical acceptable carrier. [0026] According to another aspect of the invention, it relates to use of a compound of any of the above or a pharmaceutical composition of the above in manufacturing a medicament for inhibiting capillary-like tube formation. [0027] According to another aspect of the invention, it relates to a method of inhibiting capillary-like tube formation, comprising: contacting a cell with a compound of any of the above or a pharmaceutical composition of the above. [0028] According to another aspect of the invention, it relates to a compound of formula (I), a polymorph, a tautomer, an enantiomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof for treatment of a tumor or a cancer, wherein the tumor or cancer is a bone cancer, brain and CNS tumor, breast cancer, breast cancer, colorectal cancer, endocrine cancer, gastrointestinal cancer, genitourinary cancer, gynaecological cancer, head and neck cancer, leukaemia, lung cancer, lymphoma, eye cancer, skin cancer, soft tissue sarcoma, urinary system cancer, and other types or related disorders:

(I) [0029] wherein each of R1– R6, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(O)R, OC(O)OR, OC(O)NRR', SO2R, SO3R, SO2NRR', SR, NRR', NRSO2NR'R", NRSO₂R', NRSO₃R', NRC(O)R', NRC(O)NR'R", NRC(O)OR', NRC(N)NR'R", C(O)R, C(O)OR, C(O)NRR'; and [0030] wherein each of R, R', and R", independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, cyclyl, or heterocyclyl. [0031] According to another aspect of the invention, it relates to a pharmaceutical composition for inhibiting angiogenesis, comprising a therapeutically effective amount of the compound of any of the above and a pharmaceutical acceptable carrier. [0032] The composition of the above, wherein the tumor or cancer is gastrointestinal cancer. [0033] The composition of the above, wherein the tumor or cancer is colorectal cancer. [0034] According to another aspect of the invention, it relates to use of a compound of any of the above or a pharmaceutical composition of the above in manufacturing a medicament for treatment of a tumor or a cancer, wherein the tumor or cancer is a bone cancer, brain and CNS tumor, breast cancer, breast cancer, colorectal cancer, endocrine cancer, gastrointestinal cancer, genitourinary cancer, gynaecological cancer, head and neck cancer, leukaemia, lung cancer, lymphoma, eye cancer, skin cancer, soft tissue sarcoma, urinary system cancer, and other types or related disorders. [0035] According to another aspect of the invention, it relates to a method of treatment of a tumor or a cancer, wherein the tumor or cancer is a bone cancer, brain and CNS tumor, breast cancer, breast cancer, colorectal cancer, endocrine cancer, gastrointestinal cancer, genitourinary cancer, gynaecological cancer, head and neck cancer, leukaemia, lung cancer, lymphoma, eye cancer, skin cancer, soft tissue sarcoma, urinary system cancer, and other types or related disorders, comprising: contacting a cell with a compound of any of the above or a pharmaceutical composition of the above. BRIEF DESCRIPTION OF THE DRAWINGS [0036] Hereinafter, some preferred embodiments of the invention will be described in reference to the accompanying drawings. [0037] Fig. 1a and Fig. 1b show effects of SB01, SB01-M2, Paclitaxel, and CA4 on the proliferation of five cancer cell lines; [0038] Fig. 2 shows effects of SB01, SB01-M2, Paclitaxel, and CA4 on the proliferation of AGS cells and HUVEC cells; [0039] Figs. 3a-3d show effects of SB01, SB01-M2, Paclitaxel, and CA4 on the distribution of cell cycle of AGS and HUVEC cells; [0040] Fig. 4 shows another effect SB01, SB01-M2, Paclitaxel, and CA4 on the distribution of cell cycle of AGS and HUVEC cells; [0041] Fig. 5 shows effects SB01, SB01-M2, Paclitaxel, and CA4 on the protein levels of Į -tubulin in AGS and HUVEC cells; [0042] Figs. 6a-6d show effects SB01, SB01-M2, Paclitaxel, and CA4 on the distribution and assembly of Į -tubulin in AGS and HUVEC cells; [0043] Figs. 7a-7d show another effects SB01, SB01-M2, Paclitaxel, and CA4 on the distribution and assembly of Į -tubulin in AGS and HUVEC cells; [0044] Fig. 8a and 8b show effects of SB01, SB01-M2, Paclitaxel, and CA4 on the cell morphology of AGS and HUVEC cells; [0045] Fig. 9 shows another effect of SB01, SB01-M2, Paclitaxel, and CA4 on capillary-like tube formation of HUVEC cells; [0046] Figs. 10a and 10b show another effects of SB0l, SB01-M2, Paclitaxel, and CA4 on capillary-like tube formation of HUVEC cells; [0047] Fig. 11 shows effects of SB01, SB01-M2, Paclitaxel, and CA4 on the proliferation of COLO 250 cells and HUVEC cells; [0048] Figs. 12a and 12b shows effects of SB01 and SB01-M2 on the cellular apoptosis of tumor cells in vivo; [0049] Figs. 13a and 13b shows effects of SB01 and SB01-M2 on the small vessels in tumor; [0050] Fig. 14 shows effect of SB01, SB01-M1, and SB01-M2 on the proliferation of endothelial cells; [0051] Fig. 15 shows effect of SB01, SB01-M1, and SB01-M2 on the disruption of tumor vasculature; and [0052] Fig. 16 shows effect of SB01, SB01-M1, and SB01-M2 on the cellular apoptosis of tumor cells in vivo.

DETAILED DESCRIPTION OF THE INVENTION [0053] The invention is based on further study of a VDA drug and its metabolite and the surprising findings of the study. To have the invention understood more clearly, the following definitions are provided: [0054] The“alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, and heterocyclyl” mentioned herein include both substituted and unsubstituted moieties. The term "substituted" refers to one or more substituents (which may be the same or different), each replacing a hydrogen atom. Examples of substituents include, but are not limited to, halogen, cyano, nitro, hydroxyl, amino, mercapto, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, heterocyclyl, alkyloxy, aryloxy, alksulfanyl, arylsulfanyl, alkylamino, arylamino, dialkylamino, diarylamino, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkylcarboxyl, arylcarboxyl, heteroarylcarboxyl, alkyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylcarbamido, arylcarbamido, heterocarbamido, alkylcarbamyl, arylcarbamyl, heterocarbamyl, wherein each of alkyl (including alk), alkenyl, aryl, heteroaryl, cyclyl, and heterocyclyl is optionally substituted with halogen, cyano, nitro, hydroxyl, amino, mercapto, alkyl, aryl, heteroaryl, alkyloxy, aryloxy, alkylcarbonyl, arylcarbonyl, alkylcarboxyl, arylcarboxyl, alkyloxycarbonyl, or aryloxycarbonyl. [0055] As used herein, the term "alkyl" refers to a straight-chained or branched alkyl group containing 1 to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, tert-butyl, and n-pentyl. Similarly, the term "alkenyl" or "alkynyl" refers to a straight-chained or branched alkenyl or alkynyl group containing 2 to 6 carbon atoms. The term "alkyloxyl" refers to refers to an -O-alkyl radical. [0056] The term "aryl" refers to a hydrocarbon ring system (mono-cyclic or bi-cyclic) having at least one aromatic ring. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and pyrenyl. [0057] The term "heteroaryl" refers to a hydrocarbon ring system (mono-cyclic or bi-cyclic) having at least one aromatic ring which contains at least one heteroatom such as O, N, or S as part of the ring system and the remainder being carbon. Examples of heteroaryl moieties include, but are not limited to, furyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridinyl, pyrimidinyl, quinazolinyl, and indolyl. [0058] The terms "cyclyl" and "heterocyclyl" refer to a partially or fully saturated mono-cyclic or bi-cyclic ring system having from 4 to 14 ring atoms. A heterocyclyl ring contains one or more heteroatoms (e.g., O, N, or S) as part of the ring system and the remainder being carbon. Exemplary cyclyl and heterocyclyl rings are cyclohexane, piperidine, piperazine, morpholine, thiomorpholine, and 1,4-oxazepane. [0059] The term "halogen" as used herein refers to fluorine, chlorine, bromine, and iodine. [0060] The term“pharmaceutically acceptable salt” as used herein refers to salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; (b) salts formed from elemental nions such as chlorine, bromine, and iodine, and (c) salts derived from bases, such as ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium, and salts with organic bases such as dicyclohexylamine and N-methyl-D-glucamine. [0061] As used herein, the term "therapeutically effective amount" of a compound or pharmaceutical composition refers to an amount sufficient to provide the desired therapy effect in an animal, preferably a human, suffering from a proliferative disease, such as a cancer. For example, desired therapy effect may include, without limitation, the modulation of tumor growth (e.g. tumor growth delay), tumor size, or metastasis, the reduction of toxicity and side effects associated with a particular agent, the enhancement of tumor necrosis or hypoxia, the reduction of tumor angiogenesis, the reduction of tumor re-growth, reduced o tumor retention of CEPs and other pro-angiogenic cells, the amelioration or minimization of the clinical impairment or symptoms of cancer, extending the survival of the subject beyond that which would otherwise be expected in the absence of such treatment, and the prevention of tumor growth in an animal lacking any tumor formation prior to administration, i.e., prophylactic administration. [0062] I. Compounds [0063] The compounds of the invention may be described as the following formula (I), or a polymorph, a tautomer, an enantiomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof

(I) [0064] wherein each of R₁- R₆, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(O)R, OC(O)OR, OC(O)NRR', SO2R, SO3R, SO2NRR', SR, NRR', NRSO2NR'R", NRSO2R', NRSO₃R', NRC(O)R', NRC(O)NR'R", NRC(O)OR', NRC(N)NR'R", C(O)R, C(O)OR, C(O)NRR'; and wherein each of R, R', and R", independently, is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, cyclyl, or heterocyclyl. [0065] For illustration without any limitation, a representative compound of the invention has a formula (II) below:

[0066] wherein each of R1- R6, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(O)R, OC(O)OR, OC(O)NRR', SO₂R, SO₃R, SO₂NRR', SR, NRR', NRSO₂NR'R", NRSO₂R', NRSO₃R', NRC(O)R', NRC(O)NR'R", NRC(O)OR', NRC(N)NR'R", C(O)R, C(O)OR, C(O)NRR'; and wherein each of R, R', and R", independently, is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, cyclyl, or heterocyclyl; for example, (6-methoxy-lH-indol-3-yl)-(3,4,5-trimethoxy-phenyl)-methanone, (“SB01”) when R1-R6 is hydrogen. [0067] Another representative compound has a formula (III) below:

[0068] wherein each of R1- R6, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(O)R, OC(O)OR, OC(O)NRR', SO2R, SO3R, SO2NRR', SR, NRR', NRSO2NR'R", NRSO2R', NRSO3R', NRC(O)R', NRC(O)NR'R", NRC(O)OR', NRC(N)NR'R", C(O)R, C(O)OR, C(O)NRR'; and wherein each of R, R', and R", independently, is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, cyclyl, or heterocyclyl; for example, (6-hydroxy-lH-indol-3-yl)-(3,4,5-trimethoxy-phenyl)-methanone (SB01-M2) when R₁-R₆ is hydrogen. [0069] The compounds of the invention may be prepared according to the methods disclosed in US Patent No. 7,632,955 and other methods which are available to persons having ordinary skills in the art. [0070] The compounds of the invention may be combined with a pharmaceutical acceptable diluent, excipient, or vehicle to for a pharmaceutical composition of the invention. [0071] In the manufacture of a pharmaceutical composition of the invention, the compounds of the invention, including the pharmaceutical acceptable salts thereof, are typically admixed with, inter alia, a pharmaceutical acceptable carrier, including but not limited to diluent, excipient, or vehicle. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a tablet, which may contain from 0.01 or 0.5%> to 95%> or 99% by weight of the active compound. The compounds of the invention may be combined with the carrier by any of the well-known techniques of pharmacy consisting essentially of admixing the components, optionally including one or more accessory ingredients. [0072] II. Use of Compounds and Composition [0073] The compounds of the invention exhibit apparently anti-proliferation of some kinds of cell lines of cancers and Human umbilical vein endothelial cells (HUVEC). Those compounds are useful for treatment of proliferative diseases, such as the tumors or cancers, including but not limited to a bone cancer, brain and CNS tumor, breast cancer, breast cancer, colorectal cancer, endocrine cancer, gastrointestinal cancer, genitourinary cancer, gynaecological cancer, head and neck cancer, leukaemia, lung cancer, lymphoma, eye cancer, skin cancer, soft tissue sarcoma, urinary system cancer, and other types or related disorders. In particular, some compounds of the invention surprisingly show low cytotoxic activity in comparison to most existing VDA drugs. [0074] The discovery that various cytotoxic compounds (e.g., taxanes and Vinca alkaloids), which were used as anticancer chemotherapeutic drugs previously, have been found to also have vascular targeting activities through interference with the microtubule cytoskeleton and mitotic apparatus that attracting much attention in the past years. However, these agents currently under development exhibit a narrow therapeutic window and deal with the issues of high toxicity, complex structures and syntheses, drug resistance, isolation procedures, and side effects for clinical treatment. Therefore, the compounds of the invention with a wide therapeutic window may lead to attainment of improving the issues mentioned above. [0075] Such wide therapeutic windows indicate intrinsic value as a clinically applicable anticancer therapeutic. With control of the toxicity, the compounds of the invention can be used independently or in combination with other anti-cancer or anti-tumor agents, for example agents targeting the tumor or cancer cells for treatment of the tumor or cancer of subjects. [0076] Subjects to be treated are typically human subjects although the compounds of the invention may be useful with any suitable subjects known to those skilled in the art, and particularly mammalian subjects including, in addition to humans, horses, cows, dogs, rabbits, fowl, sheep, and the like. As noted above, the present invention provides pharmaceutical composition comprising the compounds of the invention, in pharmaceutically acceptable carriers for oral, rectal, topical, buccal, parenteral, intramuscular, intradermal, or intravenous, and transdermal administration. The therapeutically effective dosage of any specific compound, the use of which is in the scope of present invention, will vary somewhat from compound to compound, patient to patient, and will depend upon the condition of the patient and the route of delivery. [0077] As a general proposition, a dosage from about 0.1 to about 100 mg/kg will have therapeutic efficacy. Toxicity concerns at the higher level may restrict dosages to a lower level such as up to about 10 mg/kg, all weights being calculated based upon the weight of the active base, including the cases where a salt is employed. Different from most existing VDA drugs, some compounds of the invention with low cytotoxic activity may employ in dosages of a high level such as over 25mg/kg, over 50mg/kg, or higher. For example, the dosage of SB01-M2 may be 25-50 mg/kg. [0078] The compounds or composition of the invention may be used for blocking blood steam. Some compounds of the invention, for example SB01-M2 could shut down the blood stream without causing cytotoxic effects and show a wide therapeutic window for being as a new anticancer drug. Accordingly, the compounds or composition of the invention may be used for blocking blood steam not only the tissue of tumor or cancer but also the non-cancer cells, such as aneurysm, arteriovenous malformation, venous malformation, lymphatic malformation, hemangioma, ischemic retinopathy, diabetic retinopathy, choroidal neovascularization, wet age-related macular degeneration and other related disorders. [0079] The compounds or composition of the invention is also useful for inhibiting polymerization of Į -tubulin. [0080] The compounds or composition of the invention may inhibit capillary-like tube formation. [0081] The present invention is explained in greater detail in the following non- limiting Examples. [0082] III. Examples [0083] Example 1: SB01 and SB01-M2 on cancel cells and HUVECs [0084] 1.1. Cell culture and cell lines [0085] Human umbilical vein endothelial cells (HUVECs) and human cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsinchu, Taiwan), or Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank, National Institute of Biomedical Innovation, Japan). HUVECs was routinely cultured in Medium 200 supplemented with Low Serum Growth Supplement (LSGS) (2% fetal bovine serum; FBS) (Invitrogen, Life Technologies, USA), as a complete component in a culture environment suitable for the growth of endothelial cells from large vessels, in a humidified atmosphere containing 5% CO2 at 37°C. Human oral squamous cell carcinoma (OSCC) cell lines HSC-3 and SAS, which belonged to head and neck carcinoma, and human ovarian adenocarcinoma cell line SK-OV-3 as well as human prostate adenocarcinoma cell line PC-3 were routinely grown to desired confluence in DMEM/F12 medium containing 10% FBS at 37°C in a humidified atmosphere with 5% CO₂. Human gastric adenocarcinoma cell line AGS was cultured in RPMI 1640 medium supplemented with 10% FBS at 37°C in a humidified atmosphere with 5% CO₂. The SB01 (DBPR104, BPROL075), SB01 metabolite M2 (SB01-M2, BPROL082), and paclitaxel were kindly provided from SynCore Biotechnology Co. Ltd (Taiwan). The Combretastatin A4 (CA4) and antibodies against actin and a-tubulin were purchased from Merck (USA). [0086] 1.2 Proliferation of cancer cells and endothelial cells [0087] The experiments were conducted to investigate the effect of SB01 and SB01-M2 against human cancer cells and HUVECs by using MTT assay as previously described in Hsu, S.C., Ou, C.C., Li, J.W., Chuang, T.C., Kuo, H.P., Liu, J.Y., Chen, C.S., Lin, S.C., Su, C.H., and Kao, M.C. (2008). Ganoderma tsugae extracts inhibit colorectal cancer cell growth via G(2)/M cell cycle arrest. J Ethnopharmacol 120, 394-401. [0088] Five cancer cell lines, including HSC-3, SAS, SK-OV-3, PC-3, and AGS were screened initially and the most drug-sensitive cancer cell line together with HUVECs were further investigated. As shown in Figure 1a and Figure 1b, four compounds SB01, SB01-M2, paclitaxel, and CA4 did not show apparent anti-proliferation effect at 72 h on four cancer lines, including HSC-3, SAS, SK-OV-3, and PC-3. The only one existence of IC50 value of SB01-M2 was up to 60 M on the SAS cancer cells. Surprisingly, the human gastric adenocarcinoma cell line AGS was the most drug-sensitive cancer line based on the initial screening tests, and the IC₅₀ values of SB01, SB01-M2, paclitaxel, and CA4 at 72 h were approximately 7.53 nM, 148.89 nM, 8.46 nM, and 5.94 nM, respectively. [0089] Subsequently, AGS and HUVEC cells were used for further time-course study at 24-, 48-, and 72-h. As shown in Table I below and Figure 1a and 1b, the 4 testing compounds showed significant inhibiting effect on the proliferation of AGS and HUVEC cells. The IC50 values of SB01, SB01-M2, paclitaxel, and CA4 for AGS at 72 h were 7.25 nM, 921.75 nM (approximately 0.9 μM), 7.70 nM, and 6.27 nM, respectively.

[0090] The discrepancy of IC50 for SB01-M2 on AGS at 72 h (i.e., 148.89 nM for SB01-M2-LD and 921.75 nM for SB01-M2-HD) might be due to the personal equation and the wide range of concentration in experimental design. For HUVECs, the IC₅₀ for the 4 testing compounds at 72 h were 8.18 nM, 6145.67 nM (approximately 6.1 uM), 53.43 nM, and 7.11 nM, respectively. These data indicated that, among the 4 testing compounds, SB01-M2 had less inhibiting effect on the proliferation of AGS (10²-10³ nM) and HUVEC ( 10³-10⁴ nM) cells, representing that SB01-M2 has lower cytotoxic activity on cells compared with the other three compounds. [0091] 1.3 Distribution of cell cycle of cancer cells and endothelial cells [0092] Further experiments were made to test the phase distribution of cell cycle of AGS and HUVECs exposed to SB01 and SB01-M2 using flow cytometry as previously described in Hsu, S.C., Ou, C.C., Li, J.W., Chuang, T.C., Kuo, H.P., Liu, J.Y., Chen, C.S., Lin, S.C., Su, C.H., and Kao, M.C. (2008). Ganoderma tsugae extracts inhibit colorectal cancer cell growth via G(2)/M cell cycle arrest. J Ethnopharmacol 120, 394-401. [0093] Cells were treated with the dosage of IC₅₀ at 72 h (obtained from Exp. 1.2) of SB01-M2 or other compounds (SB01, paclitaxel, and CA4) for 48- and 72-h. As shown in Table 2 below and Figure 3, all the 4 testing compounds apparently contributed to G2/M phase arrest and affected sub-G 1 induction of AGS and HUVEC cells at 48- and 72-h.

[0094] Interestingly, SB01 (66.93 ± 5.49% at 48 h and 68.56 ± 8.31% at 72 h) and SB01-M2 (64.32 ± 9.63% at 48 h and 61.29 ± 7.74% at 72 h at low dose as well as 71.45 ± 4.22% at 48 h and 64.33 ± 6.14% at 72 h at high dose) treatment resulted in accumulation of AGS in G2/M phase arrest accompanied with the losses from G1 phase significantly more than paclitaxel (21.71 ± 4.26% at 48 h and 22.28 ± 2.45% at 72 h) and CA4 (29.59 ± 10.91% at 48 h and 20.30 ± 3.64% at 72 h). However, all the 4 testing compounds resulted in similar effects of G1, G2/M, and sub-G 1 phase apparently in HUVEC cells. In addition, as shown in Fig. 4, SB01 and high dose of SB01-M2 contributed to G2/M phase arrest of AGS more apparent than HUVEC cells at 24 h. [0095] 1.4 Assembly of microtubule in cancer cells and endothelial cells [0096] Further analysis was made on the protein expression of Į -tubulin (a major component of microtubule) by Western blotting performed as described previously with a slight modification (see Ou, C.C., Hsu, S.C., Hsieh, Y.H., Tsou, W.L., Chuang, T.C., Liu, J.Y., and Kao, M.C. 2008). The distribution and assembly of microntbule in AGS and HUVECs were visualized by immunofluorescence assay and Images were visualized using a fluorescence microscope (see Shi, K., Jiang, Q., Li, Z., Shan, L., Li, F., An, J., Yang, Y., and Xu, C. (2013). Sodium selenite alters microtubule assembly and induces apoptosis in vitro and in vivo. J Hematol On col 6, 7.). [0097] Cells were also treated with the 4 testing compounds using the dosage of IC50 at 72 h for 48- and 72-h. The influence of protein levels of a-tubulin in AGS and HUVEC cells were tested using Western blotting as shown in Figure 5. [0098] All the 4 testing compounds did not affect the Į -tubulin level statistically in AGS cells, whereas the tendency after treating with SB01, SB01-M2 (LD and HD), and CA4 differed in change of Į -tubulin level from treating with paclitaxel; the latter resulted in an increase of the Į -tubulin level in a time-dependent manner in AGS cells. [0099] Surprisingly, treatment with SB01, SB01-M2, and CA4 in HUVEC cells showed the apparent decreases of the a-tubulin level at 48 h, and only treatment with SB01-M2 significantly decreased the Į -tubulin level until to 72 h. In addition, to determine the response of cellular microtubule networks in AGS and HUVEC cells exposed to SB01 and SB01-M2, immunostaining assay of Į -tubulin was performed. As illustrated in Figures 6a-6d (400X magnification) and Figures 7a-7d (200X magnification), the cellular microtubule networks displayed normal arrangement and assembly in AGS and HUVEC cells without the treatment of the testing compounds. However, SB01 and SB01-M2 treatment seemed to cause an inhibition of Į -tubulin polymerization (depolymerization) in AGS and HUVEC cells at 48- and 72-h similar to that of CA4-mediated microtubule networks alteration. CA4, a well-known microtubule-binding agent and a VDA, was used as a positive control showing depolymerization via binding to microtubule at the colchicine-binding site. In contrast, paclitaxel dramatically contributed to microtubule polymerization in AGS and HUVEC cells at 48- and 72-h (dashed arrows). Furthermore, we also observed that the cell morphology of HUVECs exposed to SB01, SB01-M2, paclitaxel, and CA4 were dramatically changed. The cell morphology of HUVECs was altered from spindle-shape to round-shape or even irregular-shape by SB01, SB01-M2, and CA4. Whereas the formation of thicker cell morphology was due to the microtubule polymerization caused by paclitaxel, as shown in Figures 6c and 6d, Figures 7c and 7d, and Figures 8b). In addition to the cellular microtubule networks alteration, the discovery that all the 4 testing compounds seemed to cause the mitotic catastrophe in AGS and HUVEC cells accompanied with the production of multiple micronuclei and giant cells had also attracted more attention (solid-line arrows) (Figures 6a-6d, 7a-7d, and 8b). [00100]1.5. Capillary-like tube formation of endothelial cells [00101]The capillary-like tube formation of endothelial cells is a major step in angiogenesis. According to the results obtained from Exp. 1.4, SB01 and SB01-M2 treatment resulted in inhibition of a-tubulin polymerization followed by altering cell morphology of HUVECs from spindle-shape to round-shape or even irregular-shape. HUVECs were treated with the 4 testing compounds using the dosage of IC50 at 72 h for 6 h to further verify whether SB01 and SB01-M2 could block normal physiological function of endothelial cells. The influence of SB01-M2 on the spontaneous capillary-like tube formation of HUVECs was tested using capillary-like tube formation assay. As illustrated in Figure 9 and Figures 10a and 10b, all the 4 testing compounds showed apparently inhibiting effect on the capillary-like tube formation length, whereas only treatment with SB01-M2 significantly inhibited the tube formation number of HUVECs at 4-, 6-, and 8-h exposure compared with the other three compounds. These results also indicated that SB01-M2 might be used as a potential anti-angiogenesis agent. [00102]The above experiments indicated that all compounds (SB01, SB01-M2, paclitaxel, and CA4) apparently exhibited anti-proliferation effect on AGS and HUVEC cells and also contributed to G2/M phase arrest and cell apoptosis of these two cell lines. Among the 4 testing compounds, SB01-M2 seemed to also have lower cytotoxic activity compared with the other three compounds on AGS and HUVEC cells. [00103]Furthermore, SB01 and SB01-M2 treatment seemed to decrease the Į -tubulin level in HUVEC cells more apparent than in AGS cells and caused an inhibition of Į -tubulin polymerization followed by altering cell morphology of HUVECs from spindle-shape to round-shape or even irregular-shape. Most noticeably, SB01 and SB01-M2 might lead to mitotic catastrophe accompanied with the production of giant cells and multiple micronuclei in AGS and HUVEC cells, as the other compounds of paclitaxel and CA4. [00104]Additionally, the above data also revealed that SB01 and SB01-M2 treatment resulted in interrupting the capillary-like tube formation of HUVECs, which is a major step in angiogenesis. [00105]Taken together, as to SB01-M2, the above experiments indicated that it had lower cytotoxic activity on the proliferation and cell survival of AGS and HUVEC cells but demonstrated apparent anti-microtubule activity and mitotic catastrophe, which might be the attractive mechanisms to demonstrate SB01-M2 as a new class of VDA with the shutdown capacity of blood flow and lower cytotoxic effects. [00106]More specifically, in comparison with other well-characterized clinical used anti-mitotic agents, such as paclitaxel, CA4, and even SB01, SB01-M2 had less inhibiting effect on the proliferation and survival of variety of human cancer cells, including HSC-3, SAS, SK-OV -3, and PC-3 (10-100 M and even more), as well as endothelial cells (HUVEC) (1-10 μM). Even in drug-sensitive cancer cell line (AGS), the IC50 of SB01-M2 at 72 h was up to 0.9 μM (102-103 nM) higher than paclitaxel (7.7 nM) and CA4 (6.27 nM), indicating that SB01-M2 had lower cytotoxicity compared with the other three compounds (see Table I, Figures 1a, 1b, and 2). In spite of low cytotoxicity, the mechanisms of SB01-M2 still significantly contributed to G2/M cell cycle arrest and cell apoptosis (Table 2, Figure 3a-3d and 4). Although the histogram showing only few part of sub-G1 phase was induced after treatment with SB01 and SB01-M2, it was shown that they still induced G2/M phase arrest before apoptotic cell death. [00107]The paclitaxel and CA4 are well-known microtubule-binding (anti-mitotic) agents. Paclitaxel belongs to taxanes and is a well-characterized microtubule inhibitor via inducing microtubule polymerization to stabilize microtubule. On the contrary, CA4 is a well-known microtubule-binding agent and a VDA, which causes depolymerization of microtubule via binding to microtubule at the colchicine-binding site. According to the results of immunofluorescence (Figures 6a-6d and 7a-7d), SB01 and SB01-M2 treatment led to an inhibition of Į -tubulin polymerization (depolymerization) in AGS and HUVEC cells at 48- and 72-h similar to that of CA4-mediated microtubule networks alteration. Like many anti-mitotic agents, which interfere with normal process of mitosis either by affecting microtubule stability, SB01 and SB01-M2 induced depolymerization and lead to cell cycle arrest at the G2/M phase. [00108]Interestingly, it was indicated that SB01 is a new synthetic heterocyclic analog of CA4, showing the similar effects on Į -tubulin depolymerization among SB01, SB01-M2, and CA4. However, the structure analysis of SB01 and SB01-M2 currently cannot explain the similarity between CA4 and SB01, SB01-M2. Therefore, this finding is unexpected and might indicate a new target for affecting microtubule stability. [00109]In addition, the most important discovery in the above data was that the production of mitotic catastrophe, which was characteristic of giant cell formation and multiple micronuclei production in AGS and HUVEC cells after treatment with SB0l and SB01-M2, like the other two compounds (Figure 6a-6d, 7a-7d, and 8a, 8b). Previous report has also indicated that the formation of mitotic catastrophe in endothelial cells (HUVEC) after treatment with external stimuli, such as irradiation followed by delayed programmed cell death. Furthermore, some literatures have also determined that mitotic block induced by anti-mitotic agents display different mechanisms depending on the different concentration of agents, such as paclitaxel. Treatment of SB01 and SB01-M2 surprisingly also led to this result. In addition, this finding might also be used to explain why the less part of sub-G1 phase was induced in AGS and HUVEC cells after treatment with SB01 and SB01-M2. [00110]Interestingly, the above data also confirmed that SB01 and SB01-M2 showed significantly inhibiting effect on the capillary-like tube formation length of HUVECs at 4-, 6-, and 8-h exposure (Figure 9 and 10a and 10b). Additionally, SB01-M2 could also significantly inhibit tube formation number compared with the other three compounds. These results indicated that SB01-M2 might be used as an anti-angiogenesis agent. [00111]In conclusion, this embodiment demonstrated that SB01 and SB01-M2 apparently contributing to G2/M phase arrest and anti-microtubule activity as well as the formation of mitotic catastrophe in AGS and HUVEC cells and further SB01-M2 could be a novel anti-mitotic and microtubule interrupting agent with lower cytotoxicity. These findings provide an attractive mechanism to explain the discovery of SB01-M2 as a new class of VDA with the shutdown capacity of blood flow and lower cytotoxic effects. [00112]Example 2: SB01 and SB01-M2 on HUVEC and human colorectal cancer cells [00113]2.1 Cell lines [00114]Human umbilical vein endothelial cells (HUVEC) and human colorectal cancer COLO 205 (BCRC 60054; ATCC CCL-222 TM) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HUVEC and COLO 205 were routinely cultured in Medium 200 and PRMI 1640 supplemented with 10% fetal calf serum (FCS), respectively, in a humidified atmosphere of 5% CO2 at 37°C. [00115]2.2. Proliferation of endothelial cells. [00116]The experiments were conducted to investigate the activity of these compounds against activated (growth factor-supplemented) and quiescent (growth factor-deprived) human umbilical vein endothelial cells (HUVEC) using the MTS assay. As shown in Table 3, SB01 and paclitaxel significantly suppress the proliferation of activated and quiescent HUVEC.

[00117]The median effective concentration (EC50) of SB01 (positive control) for activated HUVEC at 48 h was approximately 14.86 nM. For quiescent HUVEC, the EC₅₀ values of SB01 at 48 h was approximately 13.91 nM. The EC50 of SBM2 for both activated and quiescent HUVEC was undetectable (>1000 nM) under this experimental condition (see Table 3). These data showed that SBM2 might not inhibit proliferation of human endothelial cells. [00118]2.3. Disruption of tumor vasculature. [00119]An animal bearing solid tumor derived from colorectal cancer COLO 205 cells was evaluated to determine the efficacy of SB01 and SB01-M2 in disrupting tumor vasculature,. Tumor-implanted mice were i.v. injected with SB01-LD (25 mg/kg, n=5), SB01-HD (50 mg/kg, n=5), SBM2-LD (25 mg/kg, n=5), SBM2-HD (50 mg/kg, n=5), CA4 (50 mg/kg, n=5) and control (n=5) via the tail veins. Hoechst 33342 dye was injected into tail vein and sacrificed after 24 hours of drugs treatment. For observing the physiological morphology of the mice before sacrifice, the tumor size and weight of each mouse were measured. [00120]Responses of tumor vasculature disruption to these compounds after 24 h treatment were measured histologically by using H33342 Hoechst fluorescent dye perfusion. As shown in Figures 11, treatment with SBM2 resulted in significantly disruption of tumor vasculature in animals bearing solid tumor derived from COLO 205. Quantitative analyses of Hoechst dye staining in tumor sections indicated that SBM2 treatment caused approximately 48% vascular shutdown at 25 mg/kg and 38% vascular shutdown at 50 mg/kg as shown in Figures 11). [00121] The level of cellular apoptosis within the tumor was evaluated and it was found that SB01 and SB01-M2 may alter the solid tumor microenvironment (e.g., the level of cellular apoptosis). Responses of cellular apoptosis to SB01-M2 after 24 h treatment were measured histologically using an In Situ Cell Death Kit, POD (TUNEL, Roche). The percentage of stained nuclear to total nuclear area was calculated by using the software of NIH Image 1 and 6 photos per sample were taken randomly. As illustrated in Figures 12a and 12b, in comparison with the control group, the percentage of cellular apoptosis within the COL0205 xenografted tumors treated with 25 and 50 mg/kg of SB01 (i.v.), 50 mg/kg of SBM2 (i.v.) was increased approximately 1.37, 1.82, 1.22 times, respectively. This induction effects on cellular apoptosis were not observed in tumors treated with 25 mg/kg of SBM2 (i.v.) (0.94x) and 50 mg/kg of CA4 (i.v.) (0.83x). [00122]2.4. Amount of small vessels in tumor xenografts [00123]To identify the effect of SBM2 on endothelial cells in tumor specifically, IHC staining of CD31 was applied on tumor sections. The histological image data were further analyzed by pathologist to indicate the blood stream blockage and endothelial cell injury after test agent administration. Pathologist suggested that SBM2 treatment induced blood stream blockage obviously. Furthermore, although the vascular structure was not disrupted significantly, the endothelial cells arrangement was disrupted (Figure 13a and 13b). [00124]Proliferation and metastasis of solid tumors are dependent on the supply of nutrients and oxygen by the surrounding vasculatures. Therefore, it provides the impetus for targeting the vasculature that supports tumor growth. In fact, vascular disruption agents (VDAs), such as CA4 and ZD6126 (5), exert their influences by targeting the established tumor vasculature and causing an acute and pronounced shutdown of blood vessels, ultimately resulting in necrosis of selective tumors. However, there is still a need for discovering more effective and useful VDAs for improving cancer treatments. [00125]After experiments, the compounds of the invention (e.g., SB01 and SBM2) that exhibit a wider therapeutic window. In vitro screening assay (MTS assay) is used to determine the differentiating features of endothelial cells (HUVEC) under activated or quiescent growth conditions. Moreover, an animal bearing solid tumor derived from colorectal cancer COLO 205 cells was further established to measure the efficacy of these compounds in disrupting tumor vasculature. The data showed that SBM2 did not display marked inhibitory effect on the proliferation of HUVEC under the experimental conditions (drug concentration: 0.1-1000 nM) indicating that SBM2 does not have anti-mitotic effect in a low dosage. The effect of VDAs on shutting down blood flow in tumor can be evaluated histologically from the fluorescent intensity. In conclusion, the results show that SB01 and SB01-M2 exhibit disrupting effect on tumor vasculature. Furthermore, it also demonstrates that the solid tumor microenvironment, such as the level of cellular apoptosis within tumors, is slightly altered in COLO 205-xenografted tumors treated with SB01 and SB01-M2. Taken together, this experiment indicated that SB01 and SB01-M2 may be useful for being vascular targeting agents without cytotoxic effects. [00126]Example 3: SB01, SB01-M1, and SB01-M2 on tumor vasculature [00127]The compounds having the formula (IV) below are also representative compounds of the invention:

[00128]wherein each of R1- R6, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(O)R, OC(O)OR, OC(O)NRR', SO₂R, SO₃R, SO₂NRR', SR, NRR', NRSO₂NR'R", NRSO₂R', NRSO₃R', NRC(O)R', NRC(O)NR'R", NRC(O)OR', NRC(N)NR'R", C(O)R, C(O)OR, C(O)NRR'; and wherein each of R, R', and R", independently, is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, cyclyl, or heterocyclyl; e.g. (6-methoxyl-5-hydroxyl-1H-indol-3-yl)-(3,4,5-trimethoxy-phenyl)-methanone (SB01-M1) when R₁ and R₃-R₆ are hydrogen. [00129]3.1 Cell culture and cell lines [00130]Human umbilical vein endothelial cells (HUVEC) and human colorectal cancer COLO 205 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HUVEC and COLO 205 were routinely cultured in Medium 200 and PRMI 1640 supplemented with 10% fetal calf serum (FCS), respectively, in a humidified atmosphere of 5% CO2 at 37ć . [00131]3.2 Chemical reagents and antibodies [00132]The SB01, SB01-M1, SB01-M2, paclitaxel, Vehicle-1 for SB01 (EtOH/PEG300/Solutol, 30/20/50%, v/v/v), and Vehicle-2 for (TPGS1000/Transcutol-HP/PEG400, 20/20/60%, v/v/v) were all obtained from Sinphar Pharmaceutical Co., LTD (Taiwan). The combretastatin A-4 (CA4) was obtained from Merck (USA). The CellTiter 96® AQueous One Solution Reagent was purchased from Promega Corporation (USA). The In Situ Cell Death Detection Kit, POD was obtained from Roche (Switzerland). The H33342 Hoechst fluorescent dye was purchased from Invitrogen, Life Technologies (USA). Antibody against CD31 (ab28364) was purchased from Abcam, Inc. (USA). [00133]3.3 Endothelial cell proliferation assay [00134]Proliferation of endothelial cells was determined by using an MTS assay as previously described (4). A concentration range of 0.1 to 1,000 nM was used for testing agents (e.g., SB01, and paclitaxel). For activated growth conditions, HUVEC was seeded at a density of 9,000 cells/well onto 96-well plates in a Medium 200 containing 2% FCS. For quiescent growth conditions, HUVEC was seeded at a density of 15,000 cells/well in basal medium (Medium 200) containing 0.5% FCS. After the cells adhered onto the plate, various doses of testing agents were added to the cells, and then the cultures were incubated for 48 h. Relative cell proliferation rate was measured by using CellTiter 96^® AQueous One Solution Reagent containing the MTS according to the manufacturer’s recommendation. The final solution was measured by using a spectrophotometer at a wavelength of 492 nm. [00135]3.4. Vascular disruption assay [00136]The in vivo blood disruption experiments was performed as described previously (4). Briefly, 5 × 106 COLO 205 cells were subcutaneously implanted into the hind flank region of female BALB/c nude mice (BALB/cAnN.Cg-Foxn1nu/CrlNarl). In total, 55 mice were used for this experiment; the tumor-implanted mice were i.v. injected with SB01 (25 or 50 mg/kg), SB01-M1 (25 or 50 mg/kg), SB01-M2 (5 or 10 mg/kg), and CA4 (50 mg/kg) dissolved completely in Vehicle-1 diluted 6-fold with saline via the tail veins or orally gavaged with (50 or 100 mg/kg) suspended in Vehicle-2 solution as soon as the COLO 205-xenografted tumors were grown to an average size of 300 mm³. Twenty-four hours after treatment of these testing agents, mice were then i.v. injected with 10 mg/kg of H33342 Hoechst fluorescent dye (for blood perfusion). After 1 min, mice were sacrificed and the COLO 205-xenografted tumors were excised for other histological examinations, such as H&E staining (for measurement of necrosis), CD31 staining (for detection of endothelial cells), and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining (for measurement of apoptosis). Immunostained slides were scanned using Scan-Scope (Aperio Technologies) at 20× magnification. Quantification of H33342 Hoechst and TUNEL staining were performed using the software of NIH ImageJ (Version 1.47). [00137]3.5 Effect of SB01, SB01-M1, and SB01-M2 on proliferation of endothelial cells [00138]The activity of SB01, SB01-M1, and SB01-M2 were investigated on inhibition of the proliferation of endothelial cells against activated (growth factor-supplemented) and quiescent (growth factor-deprived) human umbilical vein endothelial cells (HUVEC) using the MTS assay. As shown in Table 4 and Supplementary Figure 14, two compounds SB01 and significantly suppress the proliferation of activated and quiescent HUVEC. Table 4: Effect of SB01, SB01-M1, and SB01-M2 on the proliferation of endothelial cells

[00139]The median effective concentration (EC₅₀) of SB01 for activated HUVEC at 48 h was approximately 14.86 nM, respectively. For quiescent HUVEC, the EC50 values of these three agents at 48 h were approximately 13.91 nM and 27.59 nM, respectively. The EC50 of SB01-M1 and SB01-M2 for both activated and quiescent HUVEC was undetectable (>1000nM) under this experimental condition. [00140]3.2. Effect of SB01, SB01-M1, and SB01-M2 on disruption of tumor vasculature [00141]An animal bearing solid tumor derived from colorectal cancer COLO 205 cells was further evaluated to determine the efficacy of SB01, SB01-M1, and SB01-M2 in disrupting tumor vasculature. [00142]Fifty-five tumor-implanted mice were i.v. injected with SB01-LD (25 mg/kg, n=5), SB01-HD (50 mg/kg, n=5), SB01-M1-LD (25 mg/kg, n=5), SB01-M1-HD (50 mg/kg, n=5), SB01-M2-LD (5 mg/kg, n=5), SB01-M2-HD (10 mg/kg, n=5), CA4 (50 mg/kg, n=5), and control (6-fold dilution of Vehicle-1, n=5) via the tail veins. Hoechst 33342 dye was injected into tail vein and sacrificed after 24 hours of drugs treatment. For observing the physiological morphology of the mice before sacrifice, the tumor sizes and weight of each mouse were measured and the results are shown in the following tables:

capability, especially in the group treated with SB01-HD. [00144]Responses of tumor vasculature disruption to these compounds after 24 h treatment were measured histologically by using H33342 Hoechst fluorescent dye perfusion. As shown in Figure 15, treatment with SB01, SB01-M1, and SB01-M2 resulted in significantly disruption of tumor vasculature in animals bearing solid tumor derived from COLO 205. Quantitative analyses of Hoechst dye staining in tumor sections indicated that SB01 treatment caused approximately 54% vascular shutdown at 25 mg/kg and 73% vascular shutdown at 50 mg/kg. Treatment with SB01-M1 caused approximately 48% vascular shutdown at 25 mg/kg and 38% vascular shutdown at 50 mg/kg. SB01-M2 treatment resulted in approximately 0% and 34% reduction in tumor vascular perfusion at 5 and 10 mg/kg, respectively. Treatment with showed up to 100% reduction in tumor vascular perfusion both at 50 and 100 mg/kg. [00145]The level of cellular apoptosis within the tumor (e.g., the level of cellular apoptosis) are evaluated to determine the solid tumor microenvironment changes after treatment of SB01, SB01-M1 and SB01-M2. Responses of cellular apoptosis to these testing compounds after 24 h treatment were measured histologically using an In Situ Cell Death Kit, POD (TUNEL, Roche). The percentage of stained nuclear to total nuclear area was calculated by using the software of NIH ImageJ and 6 photos per sample were taken randomly. As illustrated in Figure 16, in comparison with the control group, the percentage of cellular apoptosis within the COLO205 xenografted tumors treated with 25 and 50 mg/kg of SB01 (i.v.) and 50 mg/kg of SB01-M1 (i.v.), was increased approximately 1.17, 1.65, and 1.11 times, respectively. This induction effects on cellular apoptosis were not observed in tumors treated with 25 mg/kg of SB01-M1 (i.v.) (0.83×), 5 and 10 mg/kg of SB01-M2 (i.v.) (0.64× and 0.91×, respectively), and 50 mg/kg of CA4 (i.v.) (0.75×). [00146]Proliferation and metastasis of solid tumors are dependent on the supply of nutrients and oxygen by the surrounding vasculatures. Therefore, it provides the impetus for targeting the vasculature that supports tumor growth. In fact, vascular disruption agents (VDAs), such as CA4 and ZD6126 (5), exert their influences by targeting the established tumor vasculature and causing an acute and pronounced shutdown of blood vessels, ultimately resulting in necrosis of selective tumors. However, the limitation of narrow therapeutic window restricts their application in cancer treatments. The testing compounds (e.g., SB01, SB01-M1, and SB01-M2) exhibit a wider therapeutic window, in vitro screening assay (MTS assay) based on the differentiating features of endothelial cells (HUVEC) under activated or quiescent growth conditions. Moreover, an animal bearing solid tumor derived from colorectal cancer COLO 205 cells is further established to demonstrate the significant efficacy of these testing compounds in disrupting tumor vasculature. [00147]Similar to CA4, the experiments indicate that SB01 significantly inhibits the proliferation of endothelial cells (HUVEC) with no selectivity on activated or quiescent HUVEC (Table 4 and Figure 14). In addition, the data also show that the two SB01 metabolites (SB01-M1 and SB01-M2) do not display marked inhibitory effect on the proliferation of HUVEC under our experimental conditions (drug concentration: 0.1-1000 nM). The EC50 of SB01-M1 and SB01-M2 for both activated and quiescent HUVEC is undetectable (>1000 nM). Taking together with Examples 1 and 2, it is clear that these two SB01 metabolites exhibit selectivity on proliferating over confluent non-proliferating endothelial cells, which are not expected before. [00148]The effects of VDAs on shutting down blood flow in tumor have been evaluated histologically from the fluorescent intensity. According to the results of Hoechst dye staining (Figure 15), CA4 treatment (50 mg/kg) causes around 70% vascular shutdown in comparing with control groups. However, based on the experimental design and analytical methods in this example, the experimental results are much surprising. Comparing with CA4, treatment with the same dosage (50 mg/kg) of SB01 also causes about 70 % vascular shutdown and about 50% left while treating with 25 mg/kg. Although SB01-M1 and SB01-M2 are less effective in which high dose treatment cannot disrupt over 50% of the tumor vasculature, the effect of SB01-M2 at 10 mg/kg can cause about 40% tumor vascular disruption. [00149]In conclusion, the data indicate that SB01 significantly suppress the proliferation of endothelial cells (HUVEC) without exhibiting selectivity for activated or quiescent HUVEC. Treatment with SB01 also results in disruption of tumor vasculature in COLO 205-xenografted animals. The results also show that SB01-M1 and SB01-M2 exhibit disrupting effect on tumor vasculature, and moreover these two SB01 metabolites do not display marked inhibitory effect on the proliferation of HUVEC. Furthermore, it also demonstrates that the solid tumor microenvironment, such as the level of cellular apoptosis within tumors, is slightly altered in COLO 205-xenografted tumors treated with SB01 and two SB01 metabolites. Taken together, this example indicates that SB01, SB01-M1, and SB01-M2 are useful for being vascular targeting agents without concerning the side effects. [00150]The above embodiments of the invention have been disclosed for illustrative purposes and the invention is not to be limited to the particular forms or methods disclosed. Those skilled in the art will appreciate that various modifications, additions and substitutions are possible. Therefore, the invention is to cover all modifications, equivalents and alternatives falling within the scope of the appended claims.

Claims

CLAIMS We claim

1. A compound of formula (I), a polymorph, a tautomer, an enantiomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof for blocking blood stream:

wherein each of R1 - R6, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(0)R, OC(0)OR, GC(Q)NRR', SO2R, SO3R, S0₂NRR', SR, NRR', NRSO2NR'R", NRSO2R', NRSO3R', NRC(0)R', NRC(0)NR'R", NRC(0)OR', NRC(N)NR'R", C(0)R, C(0)OR, C(0)NRR^*; and

wherein each of R, R', and R", independently, is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyi, aryl, heteroaryl, cyciyl, or heterocyclyl.

2. The compound of claim 1 , wherein each of R1- R6, independently, is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyi, aryl, heteroaryl, heteroaryl, cyciyl, or heterocyclyl.

3. The compound of claim 1 , wherein R is hydrogen, halogen, or methyl.

4. The compound of claim 1 , wherein Ri-R₆, independently, is hydrogen or halogen.

5. The compound of claim 1, wherein the compound is for blocking blood stream to cells of non-cancer tissue.

6. The compound of claim 5, wherein a disorder or disease which benefits from blocking blood stream to ceils of non-cancer tissue is selected from a group consisting of aneurysm, arterio venous malformation, venous malformation, lymphatic malformation, hemangioma, ischemic retinopathy, diabetic retinopathy, choroidal neovascularization and wet age-related macular degeneration.

7. The compound of claim 1, wherein the compound is for blocking blood stream to cells of cancerous tissue.

8. The compound of claim 7, wherein a disorder or disease which benefits from blocking blood stream to cells of cancerous tissue is selected from a group consisting of bone cancer, brain and CNS tumor, breast cancer, breast cancer, colorectal cancer, endocrine cancer, gastrointestinal cancer, genitourinary cancer, gynaecological cancer, head and neck cancer, leukaemia, lung cancer, lymphoma, eye cancer, skin cancer, soft tissue sarcoma, and urinary system cancer,

9. A pharmaceutical composition for blocking blood stream, comprising a therapeutically effective amount of the compound of any of claims 1-8 and a pharmaceutical acceptable carrier.

10. The pharmaceutical composition of claim 9, wherein a therapeutically effective amount of the compound of any of claims 1-8 is between 5 mg/kg and 50 mg/kg.

11. Use of a compound of any of claims 1-8 or a pharmaceutical composition of claim 9 or 10 in manufacturing a medicament for blocking blood stream.

12. A method of blocking blood stream, comprising: contacting a cell with a compound of any of claims 1 -8 or a pharmaceutical composition of claim 9 or 10.

13. A compound of formula (I), a polymorph, a tautomer, an enantiomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof for inhibiting polymerization of a-tubulin:

wherein each of Ri - Re, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(0)R, OC(0)OR, OC(0)NR'R, SO2R, SO3R, SO2NRR', SR, NRR', NRSChNR'R", NRSO2R', NRSO3R^', NRC(0)R^', NRC(0)NR^!R", NRC(0)O'R, NRC(N)NR'R", C(0)R, C(0)OR, C(0)NRR^'; and

wherein each of R, R', and R", independently, is hydrogen, alkyl, alkenyl, alkynyi, cycloalkyl, heterocvcloalkyl, cycloalkenyl, heterocvcloalkenyl, aryl, heteroaryl, cyclyl, or heterocvclyl.

14. The compound of claim 13, wherein each of R1- R6, independently, is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloaikyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, heteroaryl, cyclyl, or heterocyclyl.

15. The compound of claim 13, wherein R is hydrogen, halogen, or methyl.

16. The compound of claim 13, wherein R 1 - Re, independently, is hydrogen or halogen.

17. A pharmaceutical composition for inhibiting polymerization of a-tubuiin, comprising a therapeutically effective amount of the compound of any of claims 13-16 and a pharmaceutical acceptable carrier.

18. The pharmaceutical composition of claim 17, wherein a therapeutically effective amount of the compound of any of claims 13- 16 is between 5 mg/kg and 50 mg/kg.

19. Use of a compound of any of claims 13-16 or a pharmaceutical composition of claim 17 or 18 in manufacturing a medicament for inhibiting polymerization of a-tubulin.

20. A method of inhibiting polymerization of a-tubulin, comprising: contacting a cell with a compound of any of claims 13-16 or a pharmaceutical composition of claim 17 or 18.

21. A compound of formula (I), a polymorph, a tautomer, an enantiomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof for inhibiting capillary-like tube formation:

(I)

wherein each of Ri - Re, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(0)R, OC(0)OR, OC(0)NRR\ SO2R, SO3R, SO2NRR', SR, NRR', NRSChNR'R", NRSO2R', NRSO3R^", NRC(0)R^', NRC(0)NR'R", NRC(0)OR\ NRC(N)NR'R", C(0)R, C(0)OR, C(0)NRR^*; and

wherein each of R, R', and R", independently, is hydrogen, alkyl, alkenyl, alkynyi, cycloalkyl, heterocycloalkyl, cycloalkenyi, heterocycloalkenyl, aryl, heteroaryl, cyclyl, or heterocyclyi.

22. The compound of claim 21, wherein each of Ri- Re, independently, is hydrogen, halogen, alkyl, alkenyl, alkynyi, cycloalkyl, heterocycloalkyl, cycloalkenyi, heterocycloalkenyl, aryl, heteroaryl, heteroaryl, cyclyl, or heterocyclyi.

23. The compound of claim 21, wherein R is hydrogen, halogen, or methyl.

24. The compound of claim 21, wherein Ri- Re, independently, is hydrogen or halogen.

25. A pharmaceutical composition for inhibiting capillary-like tube formation, comprising a therapeutically effective amount of the compound of any of claims 21-24 and a pharmaceutical acceptable carrier.

26. The pharmaceutical composition of claim 25, wherein a therapeutically effective amount of the compound of any of claims 21-24 is between 5 mg/kg and 50 mg/kg.

27. Use of a compound of any of claims 21-24 or a pharmaceutical composition of claim 25 or 26 in manufacturing a medicament for inhibiting capillary-like tube formation.

28. A method of inhibiting capillary-like tube formation, comprising: contacting a cell with a compound of any of claims 21-24 or a pharmaceutical composition of claim 25 or 26.

29. A compound of formula (I), a polymorph, a tautomer, an enantiomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof for treatment of a tumor or a cancer, wherein the (umor or cancer is a bone cancer, brain and CNS tumor, breast cancer, breast cancer, colorectal cancer, endocrine cancer, gastrointestinal cancer, genitourinary cancer, gynaecological cancer, head and neck cancer, leukaemia, lung cancer, lymphoma, eye cancer, skin cancer, soft tissue sarcoma, urinary system cancer, and other types or related disorders:

(I)

wherein each of R1 - R6, independently, is R, nitro, nitroso, cyano, azide, isothionitro, OR, OC(0)R, OC(0)OR, OC(0)NRR', SO2R, SO3R, S()₂NRR', SR., NRR, NRSCO2NR'R", NRSC)₂R^', NRSO3R', NRC(0)R', NRC(0)NR'R", NRC(0)OR', NRC(N)NR'R", C(0)R, C(O)OR, C(0)NRR'; and

wherein each of R, R', and R", independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycioalkenyl, heterocycloalkenyl, aryl, heteroaryl, cyclyl, or heterocyclyl.

30. The compound of claim 30, wherein each of R 1 - R6, independently, is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, heteroaryl, cyclyl, or heterocyclyl.

31. The compound of claim 30, wherein R is hydrogen, halogen, or methyl.

32. The compound of claim 30, wherein R1- R6, independently, is hydrogen or halogen.

33. A pharmaceutical composition for inhibiting angiogenesis, comprising a therapeutically effective amount of the compound of any of claims 30-32 and a pharmaceutical acceptable carrier.

34. The pharmaceutical composition of claim 33, wherein a therapeutically effective amount of the compound of any of claims 30-32 is between 5 mg/kg and 50 mg/kg.

35. The composition of claim 33, wherein the tumor or cancer is gastrointestinal cancer.

36. The composition of claim 33, wherein the tumor or cancer is colorectal cancer.

37. Use of a compound of any of claims 30-32 or a pharmaceutical composition of claim 33 or 34 in manufacturing a medicament for treatment of a tumor or a cancer, wherein the tumor or cancer is a bone cancer, brain and CNS tumor, breast cancer, breast cancer, colorectal cancer, endocrine cancer, gastrointestinal cancer, genitourinary cancer, gynaecological cancer, head and neck cancer, leukaemia, lung cancer, lymphoma, eye cancer, skin cancer, soft tissue sarcoma, urinary system cancer, and other types or related disorders.

38. The use of claim 37, wherein the tumor or cancer is gastrointestinal cancer.

39. The use of claim 37, wherein the tumor or cancer is colorectal cancer.

40. A method of treatment of a tumor or a cancer, wherein the tumor or cancer is a bone cancer, brain and CNS tumor, breast cancer, breast cancer, colorectal cancer, endocrine cancer, gastrointestinal cancer, genitourinary cancer, gynaecological cancer, head and neck cancer, leukaemia, lung cancer, lymphoma, eye cancer, skin cancer, soft tissue sarcoma, urinary system cancer, and other types or related disorders, comprising: contacting a cell with a compound of any of claims 30-32 or a pharmaceutical composition of claim 33 or 34.

41. The method of claim 40, wherein the tumor or cancer is gastrointestinal cancer.

42. The method of claim 40, wherein the tumor or cancer is colorectal cancer.