Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1999-12-15
2002-08-13
Rotman, Alan L. (Department: 1625)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C564S079000, C564S166000
Reexamination Certificate
active
06433187
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel compounds that are capable of covalently modifying covalently modifying &bgr;-tubulin and acting as therapeutic and diagnostic agents, and acting as therapeutic and diagnostic agents.
BACKGROUND OF THE INVENTION
Microtubules are subcellular organelles located in most eukaryotic cells and are involved in a variety of cell functions including mitosis, intracellular movement, cell movement and maintenance of cell shape. Microtubule assembly involves polymerization of tubulin and additional construction with other components of the microtubule (referred to as “microtubule-associated proteins” or MAPs).
Tubulin itself consists of two 50 kDa subunits Tubulin itself consists of two 50 kDa subunits (&agr;- and &bgr;-tubulin) which combine in a heterodimer. The heterodimer binds two molecules of guanosine triphosphate (GTP). One of the GTP molecules is tightly bound and cannot be removed without denaturing the heterodimer, while the other GTP molecule is freely exchangeable with other GTPs. This exchangeable GTP is believed to be involved in tubulin function. In particular, the tubulin heterodimer can combine in a head-to-tail arrangement in the presence of GTP to form a long protein fiber, known as a protofilament. These protofilaments can then group together to form a protein sheet which then curls into a tube-like structure known as a microtubule. Interference with this process of microtubule construction affects the downstream processes of mitosis and maintenance of cell shape.
Most of the naturally-occurring antimitotic agents have been shown to exert their effect by binding to tubulin, rather than MAPs or other proteins involved in mitosis. For example, tubulin is the biochemical target for several clinically useful anticancer drugs, including vincristine, vinblastine and paclitaxel. Another natural product, colchicine, was instrumental in the purification of tubulin as a result of its potent binding, with &bgr;-tubulin being the target for colchicine. Colchicine and other colchicine site agents bind at a site on &bgr;-tubulin that results in inhibition of a cross-link between cys-239 and cys-354 (wherein the numbering refers to the between cys-239 and cys-354 (wherein the numbering refers to the &bgr;2 isotype) by such non-specific divalent sulfhydryl reactive agents as N,N′-ethylenebis-iodoacetamide. However, simple alkylation of cys-239 does not appear to inhibit colchicine binding to tubulin.
In addition to colchicine, other natural products are known that bind at the colchicine site and inhibit microtubule assembly, for example, podophyllotoxin, steganacin and combretastatin. Still other agents bind to sites on tubulin referred to as the Vinca alkaloid site and the Rhizoxin/Maytansine site. However, none of the noted natural products are thought to operate by covalent modification of tubulin.
SUMMARY OF THE INVENTION
The present invention provides natural product derivatives as well as derivatives of known tubulin-binding compounds in which a (poly)fluorobenzene, a fluoropyridine, or a fluoronitrophenyl moiety is incorporated or added to the structure. These derivatives can be used as antimitotic agents and can be considered covalent modifiers of tubulin. The strategy developed for each of the compounds is to i) append a fluorinated electrophile (e.g., pentafluorophenylsulfonamido, 2-fluoropyridyl, or 3,5-dinitro-4-fluorophenyl) to an existing functional group in a natural product, ii) replace an aromatic ring in a natural product with a fluorinated electrophile, or iii) attach a fluorinated electrophile to an open valence in a portion of the molecule that will not interfere with recognition and binding to the tubulin site. Derivatives are provided based on colchicine, steganacin, podophyllotoxin, nocodazole, combretastatin, curacin A, vinblastine, vincristine, dolastatin, 2-methoxyestradiol, dihydroxy-pentamethoxyflavanone and others.
The present invention further provides pharmaceutical compositions containing the natural product derivatives as well as therapeutic and diagnostic methods using those compounds and compositions.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “pharmaceutically acceptable salts” is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S. M., et al, “Pharmaceutical Salts”,
Journal of Pharmaceutical Science,
1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide a compound of formula I. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme.
Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention.
The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabe
Clark David
Frankmoelle Walter
Houze Jonathan
Jaen Juan C.
Medina Julio C.
Coppins Janet
Rotman Alan L.
Tularik Inc.
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