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WO2002060374A2 - Derives de benz-1,3-azole et leurs utilisations en tant qu'inhibiteurs de l'heparanase - Google Patents

Derives de benz-1,3-azole et leurs utilisations en tant qu'inhibiteurs de l'heparanase Download PDF

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WO2002060374A2
WO2002060374A2 PCT/IL2002/000081 IL0200081W WO02060374A2 WO 2002060374 A2 WO2002060374 A2 WO 2002060374A2 IL 0200081 W IL0200081 W IL 0200081W WO 02060374 A2 WO02060374 A2 WO 02060374A2
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compound
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pharmaceutical composition
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WO2002060374A3 (fr
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Maty Ayal-Hershkovitz
Daphna Miron
Ofra Levy
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Insight Strategy And Marketing Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/18—Benzimidazoles; Hydrogenated benzimidazoles with aryl radicals directly attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D277/28—Radicals substituted by nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/84—Naphthothiazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Definitions

  • the present invention relates to heparanase inhibitors, particularly to certain benz-1,3- azoles, more particularly to benzimidazole, benzoxazole and benzothiazole derivatives, and to their use in the treatment of diseases and disorders caused by or associated with heparanase catalytic activity such as cancer, inflammatory disorders and autoimmune diseases.
  • Heparan sulfate proteoglycans are ubiquitous macromolecules associated with the cell surface and with the extracellular matrix (ECM) of various tissues. They consist of a protein core to which several linear heparan sulfate (HS) chains are covalently attached.
  • ECM extracellular matrix
  • HSPGs are also prominent components of blood vessels. In capillaries they are found mainly in the subendothelial basement membrane, where they support proliferating and migrating endothelial cells and stabilize the structure ofthe capillary wall.
  • heparanase an endo- ⁇ -D-glucuronidase that cleaves HS at specific intrachain sites
  • Heparanase released from cells removes HS molecules from the basement membrane resulting in increase of basement membrane permeability.
  • Heparanase also facilitates proteolytic degradation of the core structural components such as type IN collagen in collaboration with gelatinases.
  • blood-borne cells accomplish penetration through the basement membrane.
  • HS catabolism is observed in wound repair, inflammation, and in diabetes.
  • heparanase was found to correlate with the metastatic potential of mouse lymphoma (Nlodavsky et al., 1983), fibrosarcoma and melanoma cells (Nakajima et al., 1988). Similar correlation was observed in human breast, colon, bladder, prostate, and liver carcinomas (Nlodavsky et al., 1999). Moreover, elevated levels of heparanase were detected in sera of metastatic tumor bearing animals (Nakajima et al., 1988) and of cancer patients, in urine of highly metastatic patients (Vlodavsky et al., 1997), and in tumor biopsies (Vlodavsky et al., 1988).
  • heparanase substrates or inhibitors e.g., non- patients, in urine of highly metastatic patients (Nlodavsky et al., 1997), and in tumor biopsies (Nlodavsky et al., 1988).
  • heparanase substrates or inhibitors e.g., non-anticoagulant species of low molecular weight heparin and polysulfated saccharides
  • Heparanase is involved also in primary tumor angiogenesis. Most primary solid tumors (1-2 mm diameter) obtain their oxygen and nutrient supply through a passive diffusion from pre-existing blood vessels, however the increase in their mass beyond this size requires angiogenesis. Heparin-binding polypeptides such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are highly mitogenic for vascular endothelial cells, and are among the most potent inducers of angiogenesis. bFGF has been extracted from the subendothelial ECM produced in vitro, and from basement membranes of cornea, suggesting that ECM may serve as a reservoir for bFGF.
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • bFGF binds to HSPG in the ECM and can be released in an active form by HS-degrading enzymes. Heparanase expressed by platelets, mast cells, neutrophils, and lymphoma cells was found to be involved in the release of active bFGF from ECM and basement membranes, suggesting that heparanase activity may not only function in cell migration and invasion, but may also elicit an indirect neovascular response (Elkin et al., 2001).
  • Heparanase catalytic activity correlates with the ability of activated cells of the immune system to leave the circulation and elicit both inflammatory and autoimmune responses. Interaction of platelets, granulocytes, T and B lymphocytes, macrophages, and mast cells with the subendothelial ECM is associated with degradation of HS by heparanase (Nlodavsky et al., 1992). The enzyme is released from intracellular compartments (e.g., lysosomes, specific granules) in response to various activation signals (e.g., thrombin, calcium ionophore, immune complexes, antigens, mitogens), suggesting its regulated involvement in inflammatory sites and in autoimmune diseases.
  • various activation signals e.g., thrombin, calcium ionophore, immune complexes, antigens, mitogens
  • heparanase substrates e.g., non-anticoagulant species of low molecular weight heparin
  • EAE experimental autoimmune encephalomyelitis
  • graft rejection indicating that heparanase inhibitors may inhibit autoimmune and inflammatory diseases
  • Heparanase inhibitors have been proposed for treatment of human metastasis, for example, derivatives of siastatin B (Nishimura et al., 1994; Kawase et al., 1995), a pyran derivative isolated from the fungal strain Acremonium sp.
  • MT70646 PCT/KR00/01493
  • suramin a polysulfonated naphthylurea
  • sulfated oligosaccharides e.g., sulfated maltotetraose and maltohexaose (Parish et al., 1999)
  • sulfated polysaccharides parish et al., 1987; Lapierre et al., 1996.
  • U.S. Patent No. 5,968,822 discloses a polynucleotide encoding a polypeptide having heparanase catalytic activity and host cells, particularly insect cells, expressing said polypeptide.
  • the recombinant polypeptide having heparanase activity is said to be useful for potential treatment of several diseases and disorders such as wound healing, angiogenesis, restenosis, inflammation and neurodegenerative diseases as well as for development of new drugs that inhibit tumor cell metastasis, inflammation and autoimmunity.
  • International Patent Publication No. WO 99/57244 ofthe present applicants discloses bacterial, yeast and animal cells and methods for overexpressing recombinant heparanase in cellular systems.
  • the present invention provides, in one aspect, a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one heparanase inhibitor selected from a benz-l,3-azole ofthe general formula I hereinafter.
  • the pharmaceutical composition of the invention is particularly useful for the treatment of diseases and disorders caused by or associated with heparanase catalytic activity such as cancer, inflammatory disorders and autoimmune diseases.
  • heparanase inhibitors used in the pharmaceutical compositions of the present invention are benzimidazole, benzoxazole and benzothiazole derivatives of the general formula I hereinafter.
  • the present invention relates to the use of a benzimidazole, benzoxazole or benzothiazole derivative of the general Formula I for the manufacture of pharmaceutical compositions.
  • the compositions are for treatment of diseases and disorders caused by or associated with heparanase catalytic activity such as cancer, inflammatory disorders and autoimmune diseases.
  • the present invention provides certain novel benzimidazole, benzoxazole and benzothiazole derivatives ofthe general Formula I.
  • the present invention relates to a method for treatment of a patient suffering from a disease or disorder caused by or associated with heparanase catalytic activity such as cancer, an inflammatory disorder or an autoimmune disease, which comprises administering to said patient an effective amount of a benzimidazole, a benzoxazole or a benzothiazole derivative ofthe general Formula I.
  • compositions for treatment of diseases and disorders caused by or associated with heparanase catalytic activity, said compositions comprising a pharmaceutically acceptable carrier and at least one heparanase inhibitor which is a benz-l,3-azole ofthe general Formula I: wherein Rl is a radical selected from a radical (a)-(i) below:
  • R2 and R5 each independently represents hydrogen; halogen; -SO 3 H; C1-C6 alkoxy optionally substituted by halogen or -SO 3 H; C2-C6 alkenyl; C2-C7 alkanoyl; C1-C6 alkyl optionally substituted by halogen or C1-C6 alkoxy; C1-C6 alkylthio; or C6-C14 aryl;
  • X is NH, O or S
  • Y is a direct bond, -CH 2 -, -O-, -CO-, -SO-, -S0 2 - or -NR ⁇
  • R' is C1-C6 alkyl optionally substituted by halogen, preferably fluoro, C2-C6 alkenyl or C6-C14 aryl
  • R6 is absent or is C1-C6 alkyl or C2-C6 alkenyl, wherein said C1-C6 alkyl may optionally be substituted at the terminal carbon atom by -NR8R9 or -COOR, where R is H, C1-C6 alkyl, C2-C6 alkenyl or C6-C14 aryl;
  • R8 and R9 each independently represents hydrogen or C1-C6 alkyl; or R8 is H and R9 is C2-C7 alkanoyl or C7-C15 aroyl optionally substituted by oxo, -SO 3 H, -COOH, and/or -NH ; or the radicals R8 and R9, together with the N atom to which they are attached, form a saturated 5-7 membered heterocyclic ring optionally containing at least one further heteroatom selected from O, S and or N, said further N atom being optionally substituted by C1-C6 alkyl;
  • RIO is hydrogen; C1-C6 alkyl optionally substituted at the terminal carbon atom by -COOR wherein R is H, C1-C6 alkyl, C2-C6 alkenyl or C6-C14 aryl; Rl 1 is C1-C6 alkyl optionally substituted by fluoro; C1-C6 alkoxy; C1-C6 alkylthio; or -COOR wherein R is H, C1-C6 alkyl, C2-C6 alkenyl or C6-C14 aryl; R12 is C1-C6 alkyl or C2-C6 alkenyl; and wherein the dotted lines indicate either a double bond stretching from the carbon atom at the 2 position of the benz-l,3-azole ring to the N atom at the ring, in which case said N atom is positively charged when R6 is present, or the dotted line represents a double bond stretching from the carbon atom at the 2 position ofthe benz-l,3-azole
  • C1-C6 alkyl typically refers to a straight or branched alkyl radical having 1-6 carbon atoms and includes for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-heptyl, 2,2-dimethylpropyl, n-hexyl and the like.
  • Preferred alkyl groups are methyl and ethyl.
  • C2-C6 alkenyl refers to straight or branched hydrocarbon radicals having 2-6 carbon atoms and one, preferably a terminal, double bond, and includes for example vinyl, prop-2-en- 1 -yl, but-3-en-l-yl, pent-4-en-l-yl, and hex-5-en- 1-yl.
  • C1-C6 alkoxy refers to groups
  • alkoxy are methoxy, ethoxy, hexoxy and the like
  • examples of alkylthio are methylthio, ethylthio and propylthio, and the like
  • examples of alkanoyl are acetyl, propanoyl, butanoyl and hexanoyl.
  • C6-C14 aryl refers to an aromatic carbocyclic group having 6 to 14 carbon atoms consisting of a single ring or multiple condensed rings such as phenyl, naphthyl, and phenanthryl optionally substituted by C1-C6 alkyl.
  • C7-C15 aroyl refers to a group C6-C14 aryl-CO where aryl is as defined above and is optionally substituted by oxo, -SO 3 H, -COOH and/or -NH 2 . Examples are 9-oxofluoren-3-oyl, 2-carboxybenzoyl and 2-sulfobenzoyl.
  • heteroaryl refers to a monocyclic, bicyclic or tricyclic heteroaromatic group containing one to three heteroatoms selected from N, O and/or S such as, but not limited to, pyridyl, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, quinolinyl, thiazolyl, pyrazolyl, quinazolinyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl, isobenzofuryl, indolyl, imidazo[l ,2-a]pyridyl, benzimidazolyl, benzthiazolyl and benzoxazolyl.
  • halogen refers to fluoro, chloro, bromo or iodo.
  • the group -NR8R9 may be -NH 2 , when R8 and R9 are both hydrogen, or secondary amino when R8 is H and R9 is C1-C6 alkyl or tertiary amino when R8 and R9 are each Cl-
  • the group -NR8R9 may also be a carboxamido group when R8 is hydrogen and R9 is a C2-C7 alkanoyl or C7-C15 aroyl group as defined above, or R8 and R9 together with the nitrogen atom to which they are attached may form a saturated, preferably a 5- or 6- membered, heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from nitrogen, oxygen and/or sulfur.
  • Such rings may be substituted, for example with one or two C1-C6 alkyl groups, preferably at a further N atom.
  • rings include, without being limited to, pyrrolidino, piperidino, morpholino, thiomorpholino, diazepino, piperazino, and N-C 1 -C6 alkylpiperazino, e.g. N-methylpiperazino.
  • compositions of formula I are also contemplated by the present invention, both salts formed by any carboxy or sulfo groups present in the molecule and a base as well as acid addition and/or base salts.
  • Pharmaceutically acceptable salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.
  • metals used as cations are sodium, potassium, magnesium, calcium, and the like.
  • suitable amines are N,N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N- methylglucamine, and procaine (see, for example, Berge S. M., et al., "Pharmaceutical Salts," (1977) J. of Pharmaceutical Science, 66:1-19).
  • the salts can also be pharmaceutically acceptable quaternary salts such as a quaternary salt of the formula -NRR'R" + Z' wherein R, R'and R" each is independently hydrogen, alkyl or benzyl and Z is a counterion, including chloride, bromide, iodide, O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate.
  • quaternary salts such as a quaternary salt of the formula -NRR'R" + Z' wherein R, R'and R" each is independently hydrogen, alkyl or benzyl and Z is a counterion, including chloride, bromide, iodide, O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate.
  • Pharmaceutically acceptable acid addition salts of the compounds include salts derived from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic, phosphorous, and the like, as well as salts derived from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.
  • inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic, phosphorous, and the like
  • organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.
  • Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like.
  • salts of amino acids such as arginate and the like and gluconate or galacturonate (see, for example, Berge S. M., et al., "Pharmaceutical Salts," (1977) J. of Pharmaceutical Science, 66:1-19).
  • the acid addition salts of said basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner.
  • the free base form may be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner.
  • the free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base for purposes of the present invention.
  • the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
  • the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.
  • the free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes ofthe present invention.
  • the pharmaceutical composition comprises a compound of the general Formula I wherein Rl is a radical of the formula (a), R2, R3 and R5 are H and R4 is a radical (j), exemplified by a compound of the formula la:
  • Y may be -CH 2 - and X may be -NH-, thus obtaining a compound of the formula lb:
  • R7 is preferably -NR8R9, as identified in the formula Ic below:
  • R8 and R9 are as defined above.
  • a compound of formula Ic when -NR8R9 is -NH 2 at the para position, there is obtained the compound herein identified as Compound 1 in the Appendix A just before the Claims. This compound is described in the literature [Hamciuc et al., (1993); CAS No. 47733-85-7] but no biological activity is disclosed for it.
  • R8 is H and R9 is benzoyl substituted at the ortho position by -SO 3 H or -COOH, or R9 is a 9-oxo-fluoren-3-oyl radical, as exemplified by the novel compounds herein designated Compounds 2-4, respectively, which structural formulas are depicted in the Appendix A just before the Claims.
  • R" is preferably a quinoline residue substituted both by OH and SO 3 H such as the 5-sulfo-8-hydroxyquinolin-7-yl group, as exemplified by the novel compound herein identified as Compound 5 in the Appendix A just before the Claims.
  • the pharmaceutical composition comprises a compound of general Formula I wherein Rl is a radical of the formula (b) such as the compound ofthe formula Ie:
  • X, Y and R7 are as described hereinbefore and R3 is H and R4 is -NR8R9 or R4 is H and R3 is -NR8R9.
  • Y is O and X is NH, as is depicted in formula If:
  • R7 is -NR8R9, as depicted in formula lg:
  • the compound of formula lg -NR8R9 when in the compound of formula lg -NR8R9 is -NH 2 at the 5-position ofthe imidazole rings, the compound is herein identified as Compound 6 in the Appendix A just before the Claims.
  • This compound is described in the literature [Russian Patent Application No. 2027701 (1995); CAS No. 48229-39-6] but no biological activity is disclosed for it.
  • the novel compound is herein identified as Compound 7 in the Appendix A just before the Claims.
  • the pharmaceutical composition comprises a compound ofthe general Formula I wherein R2 and R5 are H, R6 is C1-C6 alkyl and there is a double bond between the carbon atom at position 2 and the ring N atom such as the compound of formula Ih:
  • X is S and R6 is ethyl, as depicted in the formula Ii:
  • Rl may be a radical of formula (f) as in the formula Ij:
  • RIO is ethyl, as exemplified by the compound herein designated Compound 8 in the Appendix A just before the Claims. This compound is described in the literature [CAS No. 109068-02-2] but no biological activity has been disclosed for the compound.
  • the pharmaceutical composition ofthe present invention comprises a compound of the formula Ii wherein Rl is a radical of formula (e) as in the formula II:
  • RIO is ethyl, exemplified by the compound herein designated Compound 9 in the Appendix A just before the Claims.
  • This compound is described in the literature [CAS No. 109068-03-3] but no biological activity has been disclosed for the compound.
  • the pharmaceutical composition comprises a compound ofthe formula Ii wherein Rl is a radical of formula (c), as depicted in formula In:
  • RIO is ethyl, as exemplified by the novel compound herein designated Compound 10 in the Appendix A just before the Claims.
  • the pharmaceutical composition comprises a compound ofthe general Formula I wherein R2 and R3 are hydrogen, R6 is Cl- C6 alkyl and there is a double bond between the carbon atom at position 2 and the ring N atom such as the compound of formula Ip:
  • X is S, R4 and R5 together with the carbon atoms to which they are attached form a condensed benzene ring, and R6 is ethyl, as depicted in formula Iq:
  • Rl may be a radical of formula (d), as in the compound ofthe formula Ir:
  • RIO is ethyl and Rl 1 is methyl, as exemplified by the compound herein designated Compound 11 in the Appendix A just before the Claims.
  • This compound is described in the literature [CAS No.108722-12- 9] but no biological activity has been disclosed for the compound.
  • the pharmaceutical composition comprises a compound of the general Formula I wherein R6 is C1-C6 alkyl and there is a double bond between the carbon atom at position 2 of the benz-l,3-azole ring and the first carbon atom of the Rl radical.
  • R2 and R3 are hydrogen, R4 and R5 together with the carbon atoms to which they are attached form a condensed benzene ring and Rl is a radical ofthe formula (h), as exemplified by formula Is:
  • RIO may be ethyl, as exemplified by the novel compound herein designated Compound 12 in the Appendix A just before the Claims.
  • the pharmaceutical composition of the present invention comprises a compound of the formula Iq, wherein Rl is a radical of the formula (g), as depicted in formula It:
  • RIO may be ethyl, as exemplified by the novel compound herein designated Compound 13 in the Appendix A just before the Claims.
  • the pharmaceutical composition comprises a compound of the general Formula I, wherein R4 and R5 are hydrogen, R2 and R3 together with the carbon atoms to which they are attached form a condensed benzene ring and R6 is C1-C6 alkyl, as depicted in formula Iu:
  • Rl may be a radical of the formula
  • R6 is preferably methyl, as exemplified by the compound herein designated Compound 14 in the Appendix A just before the Claims. This compound is described in the literature [CAS No. 6285-35-4] but no biological activity has been disclosed for the compound.
  • the pharmaceutical composition comprises a compound of the general Formula I, wherein Rl is a radical (a), R2 and R5 are hydrogen, R6 is absent, and R3 and R4 independently are each hydrogen or - NR8R9 as depicted in formula Iw:
  • R7 is at the para position, and R3 and R7 are -NR8R9, wherein R8 and R9 are both hydrogen, as depicted in the formula Ix:
  • X is -NH- and R4 is methyl, as exemplified by the compound herein designated Compound 15 in the Appendix A just before the Claims.
  • R7 is at the para position, R3 is H, R4 and R7 are both -NHR9, as in formula Iy:
  • X may be S
  • R9 may be benzoyl substituted at the para-position by -NH 2 , as exemplified by the compound herein designated Compound 16 in the Appendix A just before the Claims.
  • This compound is described in the literature [CAS No. 330998-38-4] but no biological activity has been disclosed for the compound.
  • R4 is H and R3 is -NH 2 , as depicted in formula Iz below:
  • R7 is benzimidazol-2-yl substituted by -
  • the present invention further relates to novel compounds ofthe general formula I and to pharmaceutically acceptable salts thereof.
  • the invention relates to a heterocyclic compound of the formula
  • R7 is preferably -NR8R9 as depicted in formula Ic:
  • the radicals -NR8R9 are at the para-positions of the phenyl rings, R8 is H and R9 is benzoyl substituted at the ortho- position by -SO 3 H or -COOH, or R9 is a 9-oxo-fluoren-3-oyl radical, as exemplified by the compounds herein designated Compounds 2-4 ofthe formulas:
  • R" is heteroaryl derived from a mono- or poly-cyclic heteroaromatic ring containing one to three heteroatoms selected from N, O and/or S and being optionally substituted by -OH, -COOH and/or -S0 3 H.
  • R" is preferably a quinoline residue substituted by both -OH and -SO 3 H such as the 5-sulfo-8-hydroxyquinolin-7-yl group, as depicted in the compound herein designated Compound 5 ofthe formula:
  • the invention relates to a heterocyclic compound of the formula lg:
  • the invention further relates to the heterocyclic compounds herein designated Compounds 10, 12 and 13 ofthe formulas:
  • the Compounds 1-7, 10, 12 and 13 ofthe invention can be prepared as illustrated in Schemes 1 to 11 in the Appendix B, herein.
  • the synthesis involves initial construction ofthe benz-l,3-azole backbone followed, if desired, by functionalization of the terminal amino groups.
  • Compound 1 was prepared in five steps from 4,4'-methylenedianiline according to Scheme 1 (depicted in the Appendix B just before the Claims).
  • step a The amino groups of 4,4'-methylenedianiline were protected by acetylation (step a), followed by nitration of the acetylated amino compound at the ortho positions with nitric acid (step b), deprotection of the amino groups with KOH (step c), reduction of the nitro groups to afford the tetra-amino compound (step d), and polyphosphoric acid mediated condensation of the tetra-amino compound with -aminobenzoic acid to give Compound 1 as a green powder.
  • Compounds 2, 3 and 4 were prepared from Compound 1 by acylation of the amino groups with the appropriate cyclic anhydride or acyl chloride, as shown in Schemes 2, 3 and 4, respectively.
  • Compound 5 was synthesized as shown in Scheme 5 by reaction of Compound 1 with sodium nitrite followed by reaction of the bis-diazonium salt with 8- hydroxyquinoline-5-sulfonic acid.
  • Compound 10, 12 and 13 were prepared in multi-step syntheses from uncharged starting materials as shown in Schemes 8-9, 10 and 11, respectively.
  • the synthesis of Compound 10 began with the Wittig reaction ofthe phosphonium salt of 1 -chloromethyl-4-methoxybenzene and 2-methylbenzothiazole-6-carbaldehyde as shown in Schemes 8 and 9.
  • Compound 12 was prepared in multiple steps from 2-methylnaphthol [2, 1 -d]thiazole, as shown in Scheme 10.
  • Compound 13 was prepared similarly to Compound 12 from 2- methylnaphtho[2,l-d]thiazole and 2-methyl-4,5-diphenyl-thiazole, as shown in Scheme 11.
  • the inhibitory effect of the compounds of the present invention on heparanase activity can be evaluated by several methods carried out in vitro, ex vivo, or in vivo. Some of the in vitro assays used according to the present invention were described in
  • the heparanase may be natural mammalian heparanase, such as human heparanase purified as described in U.S. Patent 5,362,641 or, preferably, recombinant mammalian, e.g. human or mouse recombinant heparanase as described in US 5,968,822, US 6,190,875, and WO 99/57244, in purified or non-purified form.
  • a source of non-purified recombinant heparanase is, for example, an extract of cells in which mammalian heparanase cDNA is expressed.
  • the heparanase substrate may be a natural heparan sulfate substrate, or an alternative substrate of the enzyme as described in U.S. 6,190,875, for example, heparin (e.g. heparin immobilized on a gel such as Sepharose), heparin fragments (e.g. several species of low molecular weight heparin), modified non-anticoagulant species of heparin, other sulfated polysaccharides (e.g. pentosan polysulfate), soluble HSPG or ECM. Evaluation ofthe inhibitory effect can be carried out, for example, as described in US
  • Colorimetric assays Any colorimetric assay based on any color producing reaction is envisaged by the invention, be it a simple color reaction, which is readily detectable, or a fluorimetric or a luminiscent (e.g., chemiluminiscent) reaction, which are readily detectable by fluorescence detecting techniques.
  • suitable colorimetric assays include, but are not limited to, the dimethylmethylene blue (DMB), tetrazolium blue and carbazole assays.
  • Qualitative colorimetric assays include the dimethylmethylene blue (DMB) assay, which yields color shift in the presence of polyanionic compounds such as sulfated glycosaminoglycans having different sizes that are released from the substrate (soluble or immobilized), and the carbazole assay, which detects uronic acid derivatives present in complete hydrolyzates of products released from an immobilized substrate, both assays being applicable for crude extracts of heparanase and for the purified enzyme as well.
  • DMB dimethylmethylene blue
  • a quantitative evaluation is desired and the preferred in vitro assays are those which are adapted for detection of reducing moieties associated with degradation products of the heparanase substrate, preferably a reducing sugar assay.
  • An example of a quantitative colorimetric assay is the tetrazolium blue assay which allows colorimetric detection of reducing moieties released from the substrate, e.g. heparan sulfate, which may be present either in soluble or immobilized form.
  • Another possibility consists in evaluating the catalytic activity of heparanase on the substrate by radioactive techniques, in which case the substrate used is radiolabeled, either in vitro or metabolically.
  • the ex vivo assays for evaluating the inhibitory effect of the compounds on heparanase activity include angiogenic sprout formation and transmigration assays.
  • the angiogenic sprout formation assay is carried out in the rat aorta model (Nicosia et al., 1997; Nicosia and Ottinetti, 1990), whereby rat aorta rings are embedded in a basement membranelike matrix composed of ECM-derived proteins such as laminin and collagen type IV, and HSPG, thus constituting a relevant heparanase substrate.
  • the rings then develop angiogenic sprouts and angiogenesis can be quantitated.
  • the compounds to be tested are added to the embedded aortic rings and their effect on angiogenic sprout formation is then evaluated.
  • immune cell migration is evaluated, optionally in the presence of a chemoattractant factor such as stromal cell-derived factor 1 (SDF-1), a process which mimics in vivo extravasation of immune cells from the vasculature to sites of inflammation.
  • a chemoattractant factor such as stromal cell-derived factor 1 (SDF-1)
  • SDF-1 stromal cell-derived factor 1
  • immune cells such as lymphocytes are let to migrate from the upper to the lower chamber through a transwell filter coated with a basement membrane-like matrix composed of ECM-derived proteins.
  • the migration rate of the cells through the filter is then evaluated by counting the number of cells migrated through the filter (e.g. using a FACSort) compared to the number of cells added on top of the upper chamber.
  • Over expression of heparanase in the immune cells results in an increase in the transmigration rate of the cells while addition of a heparanase inhibitor reduces the transmigration rate ofthe cells.
  • the inhibitory effect ofthe compounds on heparanase activity may be also assayed in vivo, for example, using the primary tumor growth or metastasis animal models or the sponge inflammation assay.
  • primary tumor animal model animals are injected subcutaneously (s.c.) with tumor cells and treated with the heparanase inhibitors. Tumor growth is measured when animals in untreated control group start to die.
  • primary tumors may be generated with B16-F1 melanoma cells or with a highly metastatic subclone thereof injected s.c. into the flanks of mice.
  • the mice are treated with heparanase inhibitors injected intraperitoneally (i.p.) twice a day starting 4 days after cell injection and are sacrificed and the tumor measured about 3 weeks after cell injection.
  • metastasis animal model animals are injected intravenously (i.v.) with tumor cells and treated with the heparanase inhibitors.
  • the number of lung metastasis is counted when animals in untreated control group start to die or about 3 weeks after cell injection.
  • metastasis may be generated with B16-F1 melanoma cells or with a highly metastatic subclone thereof injected i.v. to mice.
  • the mice are treated with heparanase inhibitors injected i.p. at certain times following cell injection, and are then sacrificed and the number of lung metastasis is counted.
  • polyvinyl alcohol (PVA) sponges are implanted under the mouse skin and the mouse is kept untreated or is treated with a test inhibitor agent.
  • the sponges are taken out, squeezed into a tube and the number of cells in each sample is determined.
  • the myeloperoxidase (MPO) content may be determined in a suspension of cell pellets, and the TNF- ⁇ content in the supernatant of the sample.
  • MPO myeloperoxidase
  • This assay mimics the inflammatory reaction resulting from the presence of a foreign body in the organism.
  • the heparanase inhibitors of the present invention can be used for the treatment of diseases and disorders caused by or associated with heparanase catalytic activity such as, but not limited to, cancer, inflammatory disorders and autoimmune diseases.
  • the compounds can be used for inhibition of angiogenesis, and are thus useful for the treatment of diseases and disorders associated with angiogenesis or neovascularization such as, but not limited to, tumor angiogenesis, ophthalmologic disorders such as diabetic retinopathy and macular degeneration, particularly age-related macular degeneration, reperfusion of gastric ulcer, and also for contraception or for inducing abortion at early stages of pregnancy.
  • diseases and disorders associated with angiogenesis or neovascularization such as, but not limited to, tumor angiogenesis, ophthalmologic disorders such as diabetic retinopathy and macular degeneration, particularly age-related macular degeneration, reperfusion of gastric ulcer, and also for contraception or for inducing abortion at early stages of pregnancy.
  • the compounds of general formula I are useful for treatment or inhibition of a malignant cell proliferative disease or disorder.
  • non-solid cancers e.g hematopoietic malignancies such as all types of leukemia, e.g. acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndrome (MDS), mast cell leukemia, hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphomas, Burkitt's lymphoma and multiple myeloma, as well as for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbla
  • ALL acute lymphocytic leukemia
  • AML acute myelogenous leukemia
  • the compounds of the general formula I are useful for treating or inhibiting tumors at all stages, namely tumor formation, primary tumors, tumor progression or tumor metastasis.
  • the compounds of general formula I are also useful for inhibiting or treating cell proliferative diseases or disorders such as psoriasis, hypertrophic scars, acne and sclerosis/scleroderma, and for inhibiting or treatment of other diseases or disorders such as polyps, multiple exostosis, hereditary exostosis, retrolental fibroplasia, hemangioma, and arteriovenous malformation.
  • the compounds of general formula I are useful for treatment of or amelioration of inflammatory symptoms in any disease, condition or disorder where immune and/or inflammation suppression is beneficial such as, but not limited to, treatment of or amelioration of inflammatory symptoms in the joints, musculoskeletal and connective tissue disorders, or of inflammatory symptoms associated with hypersensitivity, allergic reactions, asthma, atherosclerosis, otitis and other otorhinolaryngological diseases, dermatitis and other skin diseases, posterior and anterior uveitis, conjunctivitis, optic neuritis, scleritis and other immune and/or inflammatory ophthalmic diseases.
  • any disease, condition or disorder where immune and/or inflammation suppression is beneficial such as, but not limited to, treatment of or amelioration of inflammatory symptoms in the joints, musculoskeletal and connective tissue disorders, or of inflammatory symptoms associated with hypersensitivity, allergic reactions, asthma, atherosclerosis, otitis and other otorhinolaryngological diseases, dermatitis and other skin diseases, posterior and anterior uveit
  • the compounds of general formula I are useful for treatment of or amelioration of an autoimmune disease such as, but not limited to, Eaton- Lambert syndrome, Goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome, autoimmune hemolytic anemia (AIHA), hepatitis, insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), multiple sclerosis (MS), myasthenia gravis, plexus disorders e.g. acute brachial neuritis, polyglandular deficiency syndrome, primary biliary cirrhosis, rheumatoid arthritis, scleroderma, thrombocytopenia, thyroiditis e.g.
  • an autoimmune disease such as, but not limited to, Eaton- Lambert syndrome, Goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome, autoimmune hemolytic anemia (AIHA), hepatitis, insulin-dependent diabetes mellitus (IDDM
  • Hashimoto's disease Sj ⁇ gren's syndrome, allergic purpura, psoriasis, mixed connective tissue disease, polymyositis, dermatomyositis, vasculitis, polyarteritis nodosa, polymyalgia rheumatica, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, pemphigus, bullous pemphigoid, dermatitis herpetiformis, Crohn's disease and autism.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the carrier(s) must be acceptable in the sense that it is compatible with the other ingredients ofthe composition and are not deleterious to the recipient thereof.
  • carrier refers to a diluent, adjuvant, excipient, or any other suitable vehicle.
  • Such pharmaceutical carriers can be sterile liquids such as water and oils.
  • the pharmaceutical composition can be administered systemically, for example by parenteral, e.g. intravenous , intraperitoneal or intramuscular injection.
  • the pharmaceutical composition can be introduced to a site by any suitable route including intravenous, subcutaneous, transcutaneous, topical, intramuscular, intraarticular, subconjunctival, or mucosal, e.g. oral, intranasal, or intraocular.
  • the pharmaceutical composition is administered to the area in need of treatment. This may be achieved by, for example, local infusion during surgery, topical application, direct injection into the inflamed joint, directly onto the eye, etc.
  • the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or in solid form as tablets, capsules and the like.
  • compositions are conveniently delivered in the form of drops or aerosol sprays.
  • formulations may be presented in unit dosage form, e.g. in ampoules or in multidose containers with an added preservative.
  • compositions of the invention can also be delivered in a vesicle, in particular in liposomes.
  • the compositions can be delivered in a controlled release system.
  • the amount of the therapeutic or pharmaceutical composition of the invention which is effective in the treatment of a particular disease, condition or disorder will depend on the nature of the disease, condition or disorder and can be determined by standard clinical techniques. In general, the dosage ranges from about 0.01 mg/kg to about 50-100 mg/kg. In addition, in vitro assays as well in vivo experiments may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, condition or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For example, in order to obtain an effective mg/kg dose for humans based on data generated from mice or rat studies, the effective mg/kg dosage in mice or rats is divided by twelve or six, respectively.
  • Compound 2 was prepared from Compound 1 according to Scheme 2, as follows: Compound 1 (4.67 g, 10.86 mmol), N,N'-dimethylacetamide (150 mL) and N- methyl-morpholine (15 mL) were added to a dry reaction flask maintained under dry conditions (CaCl 2 ). The reaction mixture was cooled to 0°C in an ice bath while sulfobenzoic acid cyclic anhydride (9.35 g, 21.74 mmol) was added, thus obtaining a clear solution. The reaction mixture was left to react overnight at room temperature. Next, the solvent was removed under reduced pressure and the resulting mass was suspended in water (20 mL) at 60°C for 15 minutes.
  • Triethylamine (0.1 mL) and phthalicanhydride (14 mg, 0.09 mmol) were added to a solution of Compound 1 (10 mg, 0.02 mmol) in dry pyridine (2 mL). The reaction mixture was left to react at room temperature under dry conditions overnight. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography
  • Compound 4 was prepared from Compound 1 according to Scheme 4, as follows: A mixture of N-methylmorpholine (0.1 mL) and dimethylformamide (DMF, 92 mL) was added to a solution of Compound 1 (13 mg, 0.03 mmol) in dry pyridine (1 mL) and methylene chloride (2 mL). Next, 9-fluorenone-3-carbonyl chloride (14 mg, 0.06 mmol) was added and the slurry was left to react under dry conditions for 3 days at room temperature. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography (silica gel, CH 2 C1 2 : MeOH, 8:2), thus obtaining pure Compound 4 (13 mg., 54% yield).
  • the crude intermediate viii was refluxed in concentrated HCl (32%, 50 mL) for 2 hour. The reaction mixture was allowed to cool to room temperature and the crude intermediate ix was filtered.
  • Heparin Sepharose CL-6B was purchased from Pharmacia (Amersham Pharmacia Biotech) Uppsala, Sweden ; 1 ,9-Dimethylmethylene blue (DMB), tetrazolium blue and heparan sulfate were purchased from Sigma- Aldrich (Rehovot, Israel); MCDB 131 medium was purchased from Clonetics (San Diego, CA, USA); DMEM and fetal calf serum were purchased from Gibco BRL (InVitrogen Corporation, CA, USA) ; glutamine and gentamicin were purchased from Biological Industries (Bet Haemek, Israel). Matrigel was kindly provided by Dr. H. Kleinmann, NIDR, NIH, Bethesda, MD, USA.
  • Heparin Sepharose CL-6B beads were added up to the top of the wells of a multiscreen column loader (Millipore).
  • a 96-well multiscreen plate containing 0.65 ⁇ m hydrophilic, low protein binding, Durapore membrane (Millipore) was placed, upside down, on top of the multiscreen column loader.
  • the column loader and the multiscreen plate were held together, turned over, and the beads were uniformly transferred from the column loader to the multiscreen plate.
  • Double-distilled water (DDW) was then added to the beads, which were allowed to swell for one minute, and then washed (three times) with DDW under vacuum. Heparin concentration was estimated to be 20 ⁇ M/well.
  • WO 99/57244 was added (5 ng/well) to a reaction mixture containing 20 mM phosphate citrate buffer, pH 5.4, 1 mM CaCl 2 , 1 mM NaCl, and 1 mM dithiothreitol (DTT; total volume of 100 ⁇ l). After 3-hour incubation at 37° C in a incubator on a vortex shaker, the heparanase reaction products were filtered under vacuum and collected into a 96-well polystyrene flat bottom plate (Greiner Cat. No. 655101).
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • DMB 32 mg of DMB were dissolved in 5 ml ethanol, diluted to 1 liter with formate buffer containing 4 g sodium formate and 4 ml formic acid; 125 ⁇ l /well
  • Color was developed after 5 minutes, and the absorbance of the samples was determined using a spectrophotometer (CECIL CE2040) at 530 nm. The absorbance correlated to heparanase activity.
  • CECIL CE2040 spectrophotometer
  • heparanase was added to the heparin Sepharose swollen beads in the multiscreen plate and the heparanase reaction products were filtered immediately thereafter and the absorbance of these control samples was subtracted from all other samples.
  • crude extracts of CHO cells SI -11 subclone expressing human recombinant or crude extracts of CHO cells mhG9 clone expressing mouse recombinant heparanase (generated with the mouse heparanase cDNA as described for CHO clones expressing human recombinant heparanase in WO 99/57244) were used.
  • the cell extracts were centrifuged and resuspended in 20 mM phosphate citrate buffer, pH 5.4 containing 50 mM NaCl.
  • the cells were lysed by three cycles of freezing and thawing.
  • the cell lysates were centrifuged (lOOOOxg for 5 min), supematants were collected and then assayed for heparanase activity using the DMB assay.
  • each compound was dissolved in dimethylsulfoxide (DMSO) and added, at a concentration range of 1-30 ⁇ M, to the heparin Sepharose swollen beads in the 96- multiscreen plate.
  • DMSO dimethylsulfoxide
  • the partially purified human recombinant heparanase or the crude cell extracts expressing either human or mouse recombinant heparanase was added for a 3-hour incubation and the reaction continued as described above. Color was developed and the absorbance was measured as described above. The IC 5 o value (the concentration at which the heparanase activity was inhibited by 50%) for each compound was evaluated.
  • IC 5 o value the concentration at which the heparanase activity was inhibited by 50%
  • Human recombinant heparanase of at least 50% purity obtained by expression in the CHO cells SI -11 subclone as described in (a) above was added (4 ng) to each well of a 96- well microplate and incubated in a reaction mixture containing 20 mM phosphate citrate buffer, pH 5.4, 1 mM CaCl 2 , 1 mM NaCl, and 4 ⁇ M heparan sulfate (final volume of 100 ⁇ l).
  • the rat aorta model of angiogenesis as previously described (Nicosia et al., 1997; Nicosia and Ottinetti, 1990) was used with some modifications.
  • the rat aortic endothelium exposed to a three-dimensional matrix of collagen or other ECM-derived proteins, switches to a microvascular phenotype, generating branching networks of microvessels.
  • Angiogenesis is triggered by the injury caused by the dissection procedure and does not require stimulation by exogenous growth factors. Therefore, the rat aorta model can be used to investigate the endogenous mechanisms by which blood vessels regulate angiogenesis during wound healing.
  • thoracic aortas were excised from 2- to 3-month-old Fischer 344 male rats, rinsed in serum-free MCDB 131 growth medium containing 50 ⁇ g/ml gentamicin, cleaned of periadventitial fibroadipose tissue, and cross-sectioned at ⁇ 1 mm intervals.
  • Freshly cut aortic rings were rinsed in serum-free MCDB 131 medium and each ring was embedded in Matrigel (a basement membrane-like matrix composed of ECM-derived proteins such as laminin and collagen type IV and others, and HSPG, thus constituting a relevant heparanase substrate).
  • Matrigel a basement membrane-like matrix composed of ECM-derived proteins such as laminin and collagen type IV and others, and HSPG, thus constituting a relevant heparanase substrate.
  • Matrigel cultures were transferred to 18-mm wells of 4- well plates (Nunc) and grown at 35.5°C in 0.5 ml of serum-free MCDB 131 medium that was changed 3 times a week.
  • Angiogenesis was quantitated by counting the number of neovessels according to published criteria (Nicosia and Ottinetti, 1990).
  • a test compound was added to the Matrigel aortic ring cultures and its effect on reduction of the number of new microvessels was determined in comparison with untreated cultures.
  • primary tumor was generated in C57BL mice by cells herein designated FOR cells, which were generated as follows: B16-F1 mouse melanoma cells (ATCC No. 6326) were grown in DMEM containing 10% fetal calf serum, 2 mM glutamine, and 50 ⁇ g/ml gentamicin. A subclone ofthe B16-F1 cell line, Fl-J, produced large amounts of melanin and exhibited a highly metastasis potential. These highly metastatic Fl-J cells were injected to syngeneic mice (100,000 cells, s.c). Cells from metastases that were formed were cultured in different conditions.
  • FOR cells B16-F1 mouse melanoma cells (ATCC No. 6326) were grown in DMEM containing 10% fetal calf serum, 2 mM glutamine, and 50 ⁇ g/ml gentamicin.
  • a subclone ofthe B16-F1 cell line, Fl-J produced large amounts of melanin
  • a clone, Fl-LG, designated herein FOR was selected by its high heparanase expression and activity using the reverse transcriptase- polymerase chain reaction (RT-PCR) and the radiolabeled ECM degradation analyses, respectively, as previously described (Vlodavsky et al., 1999; U.S. 6,190,875).
  • FOR cells were grown in DMEM containing 10% fetal calf serum, 2 mM glutamine, and 50 ⁇ g/ml gentamicin until they reached confluence (typically 4-5 days) and then splitted (1 :5). This splitting yielded subconfluent and growing cells at day 7, the day of cell injection, at which the cells were trypsinized, washed with PBS and counted to yield a cell suspension of IO 6 cells/ml in PBS.
  • Male C57BL mice (-20 gram each; at least 10 mice/group) were injected s.c. on the flank with a suspension ofthe FOR cells (100 ⁇ l/mouse).
  • mice Four days later, a test compound dissolved in DMSO was injected (100 ⁇ l) i.p to the mice, twice a day (morning and evening). Each compound was injected at either 1 or 2 different concentrations (0.1 and/or 0.5 mg/mouse/day). Control mice were injected i.p. with DMSO only (100 ⁇ l). Mice were observed daily, and usually three weeks after cell injection, mice were sacrificed, the tumors were harvested and weighted. (e) In vivo mouse melanoma metastasis assay for heparanase activity
  • FOR cells were cultured as described in (d) above. After trypsinization, the cells were washed with PBS and counted to yield a cell suspension of 1.5xl0 6 cells/ml in PBS.
  • Male C57BL mice ( ⁇ 20 gram each; at least 10 mice/group) were injected i.v. with a suspension of the FOR cells (100 ⁇ l/mouse).
  • a test compound dissolved in DMSO was injected (100 ⁇ l) i.p to the mice 4 and 8 hours after cell injection. The compound was injected at 1 or 2 different concentrations ( 0.5 and or 1 mg/mouse/day). Control mice were injected i.p. with DMSO only. Mice were observed daily, and three weeks after cell injecion, mice were sacrificed, the lungs were fixed in Bouen's solution and scored for the number of metastatic nodules as previously described (Vlodavsky et al., 1995).
  • Matrigel is was composed of laminin, collagen type IV, entactin and nidogen, as well as of HSPG, thus constituting a relevant heparanase substrate.
  • the cells used in the experiment were mock- transfected Eb murine lymphoma cells not expressing heparanase and stable b/? ⁇ -transfected
  • Eb murine lymphoma cells overexpressing heparanase both cells described by Vlodavsky et al., 1999
  • the migration rate of the cells trough Matrigel was evaluated first in the absence and in the presence of the chemoattractant SDF- 1.
  • the transmigration of the Eb cells overexpressing heparanase was tested after treatment with the heparanase inhibitors of the invention. Addition of the heparanase inhibitor reduces the transmigration rate ofthe cells.
  • Example II (1). In vitro inhibition of heparanase activity by compounds of the invention
  • the inhibition of heparanase activity by the compounds of the present invention was first detected in two colorimetric in vitro assays, i.e., the DMB assay and the tetrazolium blue assay as described in Methods (a) and (b) above.
  • the human recombinant heparanase (designated h-hepa) expressed in CHO cells SI -11 subclone was used herein either in its partially purified form (50% purity) or in crude cell extracts, and the mouse recombinant heparanase (designated m-hepa) expressed in CHO cells mhG9 clone was used herein in crude cell extracts only.
  • Example II (3) Inhibition of mouse melanoma primary tumor growth by Compounds 1 and 6 and of metastasis by Compound 6 Since Compounds 1 and 6 of the present invention were shown in Example II (2) herein to inhibit angiogenic sprout formation, and since tumor progression is angiogenic- dependent, the effect of these compounds on primary tumor growth was assayed as described in Method (d) above. The results are summarized in Tables 3 and 4 for Compounds 1 and 6, respectively. As shown in Tables 3 and 4, untreated control mice developed primary tumors with an average weight of 0.1 to 0.2 g.
  • Vlodavsky I., Mohsen, M., Lider, O., Svahn, CM., Ekre, H.P., Vigoda, M., Ishai- Michaeli, R., and Peretz, T. (1994) Inhibition of tumor metastasis by heparanase inhibiting species of heparin. Invasion Metastasisl4:290-302.
  • Vlodavsky I., Ishai-Michaeli, R., Bar-Ner, M., Freidman, R., Horowitz, A.T., Fuks, Z., and Biran, S. (1988) Involvement of heparanase in tumor metastasis and angiogenesis. Isr. J. Med. 24: 464-470.
  • Vlodavsky I., Fuks, Z., Bar-Ner, M., Ariav, Y., and Schirrmacher, V. (1983) Lymphoma cell mediated degradation of sulfated proteoglycans in the subendothelial extracellular matrix: Relationship to tumor cell metastasis. Cancer Res. 43: 2704-271 1.

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Abstract

La présente invention concerne des dérivés de benz-1,3-azole, à savoir des dérivés de benzimidazole, de benzoxazole et de benzthiazole en tant qu'inhibiteurs de l'héparanase convenant pour le traitement de maladies et de troubles causés par ou associés à l'activité catalytique de l'héparanase tels que le cancer, les troubles inflammatoires et les maladies auto-immunes.
PCT/IL2002/000081 2001-01-29 2002-01-29 Derives de benz-1,3-azole et leurs utilisations en tant qu'inhibiteurs de l'heparanase WO2002060374A2 (fr)

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Cited By (25)

* Cited by examiner, † Cited by third party
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WO2004011440A1 (fr) * 2002-07-30 2004-02-05 Banyu Pharmaceutical Co., Ltd. Antagoniste de recepteur d'hormone concentrant de la melanine comprenant un derive de benzimidazole en tant qu'ingredient actif
WO2004046123A1 (fr) * 2002-11-16 2004-06-03 Oxford Glycosciences (Uk) Ltd Derives de benzoxazole, benzthiazole et benzimidazole utiles en tant qu'inhibiteurs d'heparanase
WO2004046122A3 (fr) * 2002-11-16 2004-07-15 Oxford Glycosciences Uk Ltd Derives d'acides de benzoxazole, de benzothiazole, et de benzimidazole pharmaceutiquement actifs
EP1547996A4 (fr) * 2002-08-30 2006-08-02 Bf Res Inst Inc Sondes de diagnostic et remedes contre des maladies presentant une accumulation de la proteine du prion et methode de marquage
WO2008046162A1 (fr) * 2006-10-20 2008-04-24 The Australian National University Inhibition de dégradation de matrice extracellulaire
US7514419B2 (en) 2003-11-19 2009-04-07 Metabasis Therapeutics, Inc. Phosphorus-containing thyromimetics
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WO2010017401A1 (fr) * 2008-08-07 2010-02-11 Bristol-Myers Squibb Company Inhibiteurs du virus de l’hépatite c
US7772271B2 (en) 2004-07-14 2010-08-10 Ptc Therapeutics, Inc. Methods for treating hepatitis C
US7781478B2 (en) 2004-07-14 2010-08-24 Ptc Therapeutics, Inc. Methods for treating hepatitis C
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US7868037B2 (en) 2004-07-14 2011-01-11 Ptc Therapeutics, Inc. Methods for treating hepatitis C
US8013006B2 (en) 2004-07-14 2011-09-06 Ptc Therapeutics, Inc. Methods for treating hepatitis C
JP2013527170A (ja) * 2010-05-06 2013-06-27 インコゼン セラピューティクス プライベート リミテッド 新規免疫調節剤および抗炎症化合物
CN103263414A (zh) * 2009-12-18 2013-08-28 长沙理工大学 2,2’-(1,4-亚苯基)二(苯并咪唑-5-羧酸)在制备抗肿瘤药物中的应用
US8987308B2 (en) 2010-06-01 2015-03-24 Summit Corporation Plc Compounds for the treatment of Clostridium difficile-associated disease
EP3381907A1 (fr) 2017-03-27 2018-10-03 Leadiant Biosciences SA Composés de 2-aminophényl-benzazolyl-5-acétate symétriques et leur utilisation comme anti-héparanase
WO2018177865A1 (fr) * 2017-03-27 2018-10-04 Leadiant Biosciences Sa In Liquidazione Composés destinés à être utilisés comme inhibiteurs d'héparanase
US10130643B2 (en) 2005-05-26 2018-11-20 Metabasis Therapeutics, Inc. Thyromimetics for the treatment of fatty liver diseases
US11202789B2 (en) 2016-11-21 2021-12-21 Viking Therapeutics, Inc. Method of treating glycogen storage disease
US11707472B2 (en) 2017-06-05 2023-07-25 Viking Therapeutics, Inc. Compositions for the treatment of fibrosis
US11718609B2 (en) 2016-12-13 2023-08-08 Beta Therapeutics Pty Ltd Heparanase inhibitors and use thereof
US11787828B2 (en) 2018-03-22 2023-10-17 Viking Therapeutics, Inc. Crystalline forms and methods of producing crystalline forms of a compound
US11787783B2 (en) 2016-12-13 2023-10-17 Beta Therapeutics Pty Ltd Heparanase inhibitors and use thereof
US12102646B2 (en) 2018-12-05 2024-10-01 Viking Therapeutics, Inc. Compositions for the treatment of fibrosis and inflammation

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US7541477B2 (en) 2002-07-30 2009-06-02 Banyu Pharmaceutical Co., Ltd. Antagonists to melanin-concentrating hormone receptor comprising benzimidazole derivative as active ingredient
WO2004011440A1 (fr) * 2002-07-30 2004-02-05 Banyu Pharmaceutical Co., Ltd. Antagoniste de recepteur d'hormone concentrant de la melanine comprenant un derive de benzimidazole en tant qu'ingredient actif
EP1547996A4 (fr) * 2002-08-30 2006-08-02 Bf Res Inst Inc Sondes de diagnostic et remedes contre des maladies presentant une accumulation de la proteine du prion et methode de marquage
WO2004046122A3 (fr) * 2002-11-16 2004-07-15 Oxford Glycosciences Uk Ltd Derives d'acides de benzoxazole, de benzothiazole, et de benzimidazole pharmaceutiquement actifs
WO2004046123A1 (fr) * 2002-11-16 2004-06-03 Oxford Glycosciences (Uk) Ltd Derives de benzoxazole, benzthiazole et benzimidazole utiles en tant qu'inhibiteurs d'heparanase
US7514419B2 (en) 2003-11-19 2009-04-07 Metabasis Therapeutics, Inc. Phosphorus-containing thyromimetics
US7829552B2 (en) 2003-11-19 2010-11-09 Metabasis Therapeutics, Inc. Phosphorus-containing thyromimetics
US8013006B2 (en) 2004-07-14 2011-09-06 Ptc Therapeutics, Inc. Methods for treating hepatitis C
US7772271B2 (en) 2004-07-14 2010-08-10 Ptc Therapeutics, Inc. Methods for treating hepatitis C
US7781478B2 (en) 2004-07-14 2010-08-24 Ptc Therapeutics, Inc. Methods for treating hepatitis C
US7868037B2 (en) 2004-07-14 2011-01-11 Ptc Therapeutics, Inc. Methods for treating hepatitis C
US7973069B2 (en) 2004-07-14 2011-07-05 Ptc Therapeutics, Inc. Methods for treating hepatitis C
US7645881B2 (en) 2004-07-22 2010-01-12 Ptc Therapeutics, Inc. Methods for treating hepatitis C
US10925885B2 (en) 2005-05-26 2021-02-23 Metabasis Therapeutics, Inc. Thyromimetics for the treatment of fatty liver diseases
US10130643B2 (en) 2005-05-26 2018-11-20 Metabasis Therapeutics, Inc. Thyromimetics for the treatment of fatty liver diseases
WO2008046162A1 (fr) * 2006-10-20 2008-04-24 The Australian National University Inhibition de dégradation de matrice extracellulaire
CN102177141A (zh) * 2008-08-07 2011-09-07 百时美施贵宝公司 作为丙型肝炎病毒抑制剂的联-1h-苯并咪唑
JP2011530531A (ja) * 2008-08-07 2011-12-22 ブリストル−マイヤーズ スクイブ カンパニー Ｃ型肝炎ウイルス阻害剤としてのｂｉ−１ｈ−ベンズアミダゾール
WO2010017401A1 (fr) * 2008-08-07 2010-02-11 Bristol-Myers Squibb Company Inhibiteurs du virus de l’hépatite c
WO2010063996A3 (fr) * 2008-12-02 2010-12-29 Summit Corporation Plc Composés antibactériens
JP2012510458A (ja) * 2008-12-02 2012-05-10 サミット コーポレイション ピーエルシー 抗細菌化合物
AU2009323854B2 (en) * 2008-12-02 2014-06-26 Summit (Oxford) Limited Antibacterial compounds
US8975416B2 (en) 2008-12-02 2015-03-10 Summit Corporation Plc Antibacterial compounds
EP3095783A1 (fr) * 2008-12-02 2016-11-23 Summit Corporation Plc Composés antibactériens
US9763925B2 (en) 2008-12-02 2017-09-19 Summit Therapeutics Plc Antibacterial compounds
US9314456B2 (en) 2008-12-02 2016-04-19 Summit Therapeutics Plc Antibacterial compounds
CN103263414A (zh) * 2009-12-18 2013-08-28 长沙理工大学 2,2’-(1,4-亚苯基)二(苯并咪唑-5-羧酸)在制备抗肿瘤药物中的应用
JP2013527170A (ja) * 2010-05-06 2013-06-27 インコゼン セラピューティクス プライベート リミテッド 新規免疫調節剤および抗炎症化合物
US9278091B2 (en) 2010-06-01 2016-03-08 Summit Therapeutics Plc Compounds for the treatment of Clostridium difficile associated disease
US8987308B2 (en) 2010-06-01 2015-03-24 Summit Corporation Plc Compounds for the treatment of Clostridium difficile-associated disease
US11202789B2 (en) 2016-11-21 2021-12-21 Viking Therapeutics, Inc. Method of treating glycogen storage disease
US11787783B2 (en) 2016-12-13 2023-10-17 Beta Therapeutics Pty Ltd Heparanase inhibitors and use thereof
US11718609B2 (en) 2016-12-13 2023-08-08 Beta Therapeutics Pty Ltd Heparanase inhibitors and use thereof
WO2018177865A1 (fr) * 2017-03-27 2018-10-04 Leadiant Biosciences Sa In Liquidazione Composés destinés à être utilisés comme inhibiteurs d'héparanase
WO2018177857A1 (fr) 2017-03-27 2018-10-04 Leadiant Biosciences Sa In Liquidazione Composés symétriques de 2-aminophényl-benzazolyl-5-acétate et leur utilisation comme anti-héparanase
EP3381907A1 (fr) 2017-03-27 2018-10-03 Leadiant Biosciences SA Composés de 2-aminophényl-benzazolyl-5-acétate symétriques et leur utilisation comme anti-héparanase
US11707472B2 (en) 2017-06-05 2023-07-25 Viking Therapeutics, Inc. Compositions for the treatment of fibrosis
US11787828B2 (en) 2018-03-22 2023-10-17 Viking Therapeutics, Inc. Crystalline forms and methods of producing crystalline forms of a compound
US12227533B2 (en) 2018-03-22 2025-02-18 Viking Therapeutics, Inc. Crystalline forms and methods of producing crystalline forms of a compound
US12102646B2 (en) 2018-12-05 2024-10-01 Viking Therapeutics, Inc. Compositions for the treatment of fibrosis and inflammation

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