Patent Application
20090036429
The present invention relates to chemical agents affecting levels of gene expression in cellular systems, including cancer cells, as well as the activity of polypeptides, especially those integral to cellular processes, including those encoded by said gene expression. In particular, the present invention relates to derivatives of a hydroxypiperidine moiety, and similar ring structures, processes for their preparation, their use as antitumor drugs and pharmaceutical compositions containing these drugs as active ingredients.
Screening assays for novel drugs are based on the response of model cell based systems in vitro to treatment with specific compounds. Various measures of cellular response have been utilized, including the release of cytokines, alterations in cell surface markers, activation of specific enzymes, as well as alterations in ion flux and/or pH. Some such screens rely on specific genes, such as oncogenes or tumor suppressors.
The present invention utilizes screening of small molecule compounds as potential anticancer drugs by taking advantage of the concept that for each specific tumor type, a unique signature set of genes, that are differentially expressed in tumor cells if compared to corresponding normal cells, can be established. The relatively small signature set, containing 10-30 genes, allows for easy, high throughput screening for compounds that can reverse the gene expression profile from patterns typical for cancer cells to patterns seen in normal cells. As a part of our efforts to provide new diversified compounds for high throughput gene expression screening, we designed and synthesized a number of novel derivatives of hydroxypiperidines. Gene expression screening and subsequent cytotoxicity screening revealed that some of the compounds possess biological activity. Consequently, a detailed structure-activity study relationship resulted in compounds of formula I as new small molecule agents having antineoplastic activity.
In one aspect, the present invention relates to organic compounds, derivatives of hydroxypiperidine, that have the ability to function as modulators, either inhibitors or agonists, of biological molecules, especially proteins and polypeptides, found in cells and whose function, whether normal or aberrant, is associated, either intimately or peripherally, with the cancerous process. Such compounds may operate to modulate proteins and polypeptides found inside cells, in culture or in an animal, preferably a mammal, most preferably a human being, or may operate on such proteins and polypeptides outside cells, such as in the plasma or other tissues of said animal. In general, the mechanism of action of said compounds is not essential to the functioning of the present invention and such compounds are disclosed herein without limitation as to such mechanisms. In addition, the proteins and/or polypeptides that are the targets of such compounds include those that function as enzymes, such as proteases or other metabolic constituents, or that function as structural or constitutive proteins, and said target may also include oligopeptides involved in the cancerous process.
In another aspect, the present invention relates to organic compounds, derivatives of hydroxypiperidine, that have the ability to function as gene expression modulators for genes found in cancer cells, especially genes involved in misregulated signal transduction pathways typical for colon cancer.
In one embodiment of the present invention, the compounds disclosed herein are able to up regulate genes found to be up regulated in normal (i.e., non-cancerous) cells versus cancer cells, especially colon cancer cells, thereby producing an expression profile for said gene(s) that resembles the expression profile found in normal cells. In another embodiment, the compounds disclosed herein are found to down regulate genes otherwise up-regulated in cancer cells, especially colon cancer cells, relative to normal (i.e., non-cancerous) cells thereby producing an expression profile for said gene(s) that more resembles the expression profile found in normal cells. Thus, in addition to activity in modulating a particular gene that may or may not have a major role in inducing or sustaining a cancerous condition, the agents disclosed herein also find value in regulating a set of genes whose combined activity is related to a disease condition, such as cancer, especially colon cancer, including adenocarcinoma of the colon. Thus, while an overall set of genes is modulated, the effect of modulating any subset of these may be disproportionately large or small with respect to the effect in ameliorating the overall disease process. Consequently, different disease conditions may rely on different subsets of genes to be active or inactive as a basis for the overall disease process.
Thus, the present invention relates to novel organic compounds that have the ability to function as gene modulators for genes found in normal (i.e., non-cancer) cells and which genes are found to be up regulated or down regulated in normal cells, especially colon cells. Such an effect may prevent a disease condition, such as cancer, from arising in those otherwise more susceptible to such a condition. In one such embodiment, administration of one or more of the agents disclosed herein may succeed in preventing a cancerous condition from arising.
In other embodiments, the agents disclosed herein find use in combination with each other as well as with other agents, such as where a mixture of one or more of the agents of the present invention are given in combination or where one or more of the agents disclosed herein is given together with some other already known therapeutic agent, possibly as a means of potentiating the affects of such known therapeutic agent or vice versa.
The present invention also relates to processes of preventing or treating disease conditions, especially cancer, most especially colon cancer, by administering to a subject, such as a mammal, especially a human, a therapeutically active amount of one or more of the agents disclosed herein, including where such agents are given in combination with one or more known therapeutic agents.
The following is a list of definitions for terms used herein.
“Acyl” or “carbonyl” is a radical formed by removal of the hydroxy from a carboxylic acid (i.e., R—C(═O)—). Preferred acyl groups include (for example) acetyl, formyl, and propionyl.
“Alkyl” is a saturated hydrocarbon chain having 1 to 15 carbon atoms, preferably 1 to 10, more preferably 1 to 5 carbon atoms and most preferably 1 to 4 carbon atoms. “Alkenyl” is a hydrocarbon chain having at least one (preferably only one) carbon-carbon double bond and having 2 to 15 carbon atoms, preferably 2 to 10, more preferably 2 to 5, most preferably 2 to 4 carbon atoms. “Alkynyl” is a hydrocarbon chain having at least one (preferably only one) carbon-carbon triple bond and having 2 to 15 carbon atoms, preferably 2 to 10, more preferably 2 to 4 carbon atoms. Alkyl, alkenyl and alkynyl chains (referred to collectively as “hydrocarbon chains”) may be straight or branched and may be unsubstituted or substituted. Preferred branched alkyl, alkenyl and alkynyl chains have one or two branches, preferably one branch. Preferred chains are alkyl. Alkyl, alkenyl and alkynyl hydrocarbon chains each may be unsubstituted or substituted with from 1 to 4 substituents; when substituted, preferred chains are mono-, di-, or tri-substituted. Alkyl, alkenyl and alkynyl hydrocarbon chains each may be substituted with halo, hydroxy, aryloxy (e.g., phenoxy), heteroaryloxy, acyloxy (e.g., acetoxy), carboxy, aryl (e.g., phenyl), heteroaryl, cycloalkyl, heterocycloalkyl, spirocyclic substituents, amino, amido, acylamino, keto, thioketo, cyano, or any combination thereof. Preferred hydrocarbon groups include methyl, ethyl, propyl, isopropyl, butyl, vinyl, allyl, butenyl, and exomethylenyl.
Also, as referred to herein, a “lower” alkyl, alkene or alkyne moiety (e.g., “lower alkyl”) is a chain comprised of 1 to 6, preferably from 1 to 4, carbon atoms in the case of alkyl and 2 to 6, preferably 2 to 4, carbon atoms in the case of alkene and alkyne.
“Alkoxy” is an oxygen radical having a hydrocarbon chain substituent, where the hydrocarbon chain is an alkyl or alkenyl (i.e., —O-alkyl or —O-alkenyl). Preferred alkoxy groups include (for example) methoxy, ethoxy, propoxy and allyloxy.
“Aryl” is an aromatic hydrocarbon ring. Aryl rings are monocyclic or fused bicyclic and tricyclic ring systems. Monocyclic aryl rings contain 6 carbon atoms in the ring. Monocyclic aryl rings are also referred to as phenyl rings. Bicyclic aryl rings contain from 8 to 17 carbon atoms, preferably 9 to 12 carbon atoms, in the ring. Bicyclic aryl rings include ring systems wherein one ring is aryl and the other ring is aryl, cycloalkyl, or heterocycloakyl. Preferred bicyclic aryl rings comprise 5-, 6- or 7-membered rings fused to 5-, 6-, or 7-membered rings. Aryl rings may be unsubstituted or substituted with from 1 to 4 substituents on the ring. Aryl may be substituted with halo, cyano, nitro, hydroxy, carboxy, amino, acylamino, alkyl, heteroalkyl, haloalkyl, phenyl, aryloxy, alkoxy, heteroalkyloxy, carbamyl, haloalkyl, methylenedioxy, heteroaryloxy, or any combination thereof. Preferred aryl rings include naphthyl, tolyl, xylyl, and phenyl. The most preferred aryl ring radical is phenyl.
“Alkylaryl” or “alkaryl” is an aryl ring having an alkyl group attached thereto as a substituent, wherein the alkyl is as already defined and the aryl ring may be substituted or unsubstituted. The alkyl moiety may be single or branched chain, substituted or unsubstituted.
“Arylalkyl” or “aralkyl” is an alkyl group as defined herein with an aryl ring attached thereto as a substituent and wherein the alkyl may be straight or branched and may be substituted or unsubstituted.
“Aryloxy” is an oxygen radical having an aryl substituent (i.e., —O-aryl). Preferred aryloxy groups include (for example) phenoxy, napthyloxy, methoxyphenoxy, and methylenedioxyphenoxy.
“Cycloalkyl” is a saturated or unsaturated hydrocarbon ring. Cycloalkyl rings are not aromatic. Cycloalkyl rings are monocyclic, or are fused, spiro, or bridged bicyclic ring systems. Monocyclic cycloalkyl rings contain from about 3 to about 9 carbon atoms, preferably from 3 to 7 carbon atoms, in the ring. Bicyclic cycloalkyl rings contain from 7 to 17 carbon atoms, preferably from 7 to 12 carbon atoms, in the ring. Preferred bicyclic cycloalkyl rings comprise 4-, 5-, 6- or 7-membered rings fused to 5-, 6-, or 7-membered rings. Cycloalkyl rings may be unsubstituted or substituted with from 1 to 4 substituents on the ring. Cycloalkyl may be substituted with halo, cyano, alkyl, heteroalkyl, haloalkyl, phenyl, keto, hydroxy, carboxy, amino, acylamino, aryloxy, heteroaryloxy, or any combination thereof. Preferred cycloalkyl rings include cyclopropyl, cyclopentyl, and cyclohexyl.
“Halo” or “halogen” is fluoro, chloro, bromo or iodo. Preferred halo are fluoro, chloro and bromo; more preferred typically are chloro and fluoro, especially fluoro.
“Haloalkyl” is a straight, branched, or cyclic hydrocarbon substituted with one or more halo substituents. Preferred are C1-C12 haloalkyls; more preferred are C1-C6 haloalkyls; still more preferred still are C1-C3 haloalkyls. Preferred halo substituents are fluoro and chloro. The most preferred haloalkyl is trifluoromethyl.
“Heteroatom” is a nitrogen, sulfur, or oxygen atom. Groups containing more than one heteroatom may contain different heteroatoms.
“Heteroalkyl” is a saturated or unsaturated chain containing carbon and at least one heteroatom, wherein no two heteroatoms are adjacent. Heteroalkyl chains contain from 2 to 15 member atoms (carbon and heteroatoms) in the chain, preferably 2 to 10, more preferably 2 to 5. For example, alkoxy (i.e., —O-alkyl or —O-heteroalkyl) radicals are included in heteroalkyl. Heteroalkyl chains may be straight or branched. Preferred branched heteroalkyl chains have one or two branches, preferably one branch. Preferred heteroalkyl chains are saturated. Unsaturated heteroalkyl chains have one or more carbon-carbon double bonds and/or one or more carbon-carbon triple bonds. Preferred unsaturated heteroalkyl chains have one or two double bonds or one triple bond, more preferably one double bond. Heteroalkyl chains may be unsubstituted or substituted with from 1 to 4 substituents. Preferred substituted heteroalkyl chains are mono-, di-, or tri-substituted. Heteroalkyl chains may be substituted with lower alkyl, haloalkyl, halo, hydroxy, aryloxy, heteroaryloxy, acyloxy, carboxy, monocyclic aryl, heteroaryl, cycloalkyl, heterocycloalkyl, spirocyclic substituents, amino, acylamino, amido, keto, thioketo, cyano, or any combination thereof.
“Heteroaryl” is an aromatic ring containing carbon atoms and from 1 to about 6 heteroatoms in the ring. Heteroaryl rings are monocyclic or fused bicyclic ring systems. Monocyclic heteroaryl rings contain from about 5 to about 9 member atoms (carbon and heteroatoms), preferably 5 or 6 member atoms, in the ring. Bicyclic heteroaryl rings contain from 8 to 17 member atoms, preferably 8 to 12 member atoms, in the ring. Bicyclic heteroaryl rings include ring systems wherein one ring is heteroaryl and the other ring is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl. Preferred bicyclic heteroaryl ring systems comprise 5-, 6- or 7-membered rings fused to 5-, 6-, or 7-membered rings. Heteroaryl rings may be unsubstituted or substituted with from 1 to 4 substituents on the ring. Heteroaryl may be substituted with halo, cyano, nitro, hydroxy, carboxy, amino, acylamino, alkyl, heteroalkyl, haloalkyl, phenyl, alkoxy, aryloxy, heteroaryloxy, or any combination thereof. Preferred heteroaryl rings include, but are not limited to, the following:
“Heteroaryloxy” is an oxygen radical having a heteroaryl substituent (i.e., —O-heteroaryl). Preferred heteroaryloxy groups include (for example) pyridyloxy, furanyloxy, (thiophene)oxy, (oxazole)oxy, (thiazole)oxy, (isoxazole)oxy, pyrmidinyloxy, pyrazinyloxy, and benzothiazolyloxy.
“Heterocycloalkyl” is a saturated or unsaturated ring containing carbon atoms and from 1 to about 4 (preferably 1 to 3) heteroatoms in the ring. Heterocycloalkyl rings are not aromatic. Heterocycloalkyl rings are monocyclic, or are fused, bridged, or spiro bicyclic ring systems. Monocyclic heterocycloalkyl rings contain from about 3 to about 9 member atoms (carbon and heteroatoms), preferably from 5 to 7 member atoms, in the ring. Bicyclic heterocycloalkyl rings contain from 7 to 17 member atoms, preferably 7 to 12 member atoms, in the ring. Bicyclic heterocycloalkyl rings contain from about 7 to about 17 ring atoms, preferably from 7 to 12 ring atoms. Bicyclic heterocycloalkyl rings may be fused, Spiro, or bridged ring systems. Preferred bicyclic heterocycloalkyl rings comprise 5-, 6- or 7-membered rings fused to 5-, 6-, or 7-membered rings. Heterocycloalkyl rings may be unsubstituted or substituted with from 1 to 4 substituents on the ring. Heterocycloalkyl may be substituted with halo, cyano, hydroxy, carboxy, keto, thioketo, amino, acylamino, acyl, amido, alkyl, heteroalkyl, haloalkyl, phenyl, alkoxy, aryloxy or any combination thereof. Preferred substituents on heterocycloalkyl include halo and haloalkyl. Preferred heterocycloalkyl rings include, but are not limited to, the following:
A “pharmaceutically-acceptable salt” is a cationic salt formed at any acidic (e.g., carboxylic acid) group, or an anionic salt formed at any basic (e.g., amino) group. Many such salts are known in the art, as described in World Patent Publication 87/05297, Johnston et al., published Sep. 11, 1987 incorporated by reference herein. Preferred cationic salts include the alkali metal salts (such as sodium and potassium), and alkaline earth metal salts (such as magnesium and calcium) and organic salts. Preferred anionic salts include the halides (such as chloride salts), sulfonates, carboxylates, phosphates, and the like.
Such salts are well understood by the skilled artisan, and the skilled artisan is able to prepare any number of salts given the knowledge in the art. Furthermore, it is recognized that the skilled artisan may prefer one salt over another for reasons of solubility, stability, formulation ease and the like. Determination and optimization of such salts is within the purview of the skilled artisan's practice.
A “solvate” is a complex formed by the combination of a solute (e.g., a metalloprotease inhibitor) and a solvent (e.g., water). See J. Honig et al., The Van Nostrand Chemist's Dictionary, p. 650 (1953). Pharmaceutically acceptable solvents used according to this invention include those that do not interfere with the biological activity of the metalloprotease inhibitor (e.g., water, ethanol, acetic acid, N,N-dimethylformamide and others known or readily determined by the skilled artisan). When the solvate is water it is a hydrate.
The terms “optical isomer”, “stereoisomer”, and “diastereomer” have the accepted meanings (see, e.g., Hawley's Condensed Chemical Dictionary, 11th Ed.). The illustration of specific protected forms and other derivatives of the compounds of the instant invention is not intended to be limiting. The application of other useful protecting groups, salt forms, etc. is within the ability of the skilled artisan.
The term “metabolite” refers to a product formed from a compound of the invention by ordinary physiological processes, such as enzymatic metabolism following administration of the compound of the invention to an animal, and includes a product formed by a “prodrug” which is a chemical entity that can form a compound of the invention when administered to an animal and is then subjected to normal enzymatic and/or metabolic reactions, usually but not always catalyzed by an enzyme or by stomach acids.
Where the description of substituents for more than one substituent (i.e., more than one R group) recites that said groups are “selected independently” or are “independently selected” this means that the two or more R groups may be either the same or different from each other.
In one aspect, the present invention relates to a compound having, in general, the structure of Formula I, Formula II, Formula III, Formula IV, Formula V, and/or Formula VI:
wherein
including all pharmaceutically acceptable salts, derivatives, prodrugs, metabolites, solvates, hydrates, and isomers thereof.
In a preferred embodiment of the compounds of Formula I, n=2. In other preferred embodiments, m=2. In yet other preferred embodiments, R9 is H, Cl or OMe. In still other preferred embodiments thereof, when NR13(CH2)nR14 is piperazine the ring N not attached to the C═O may be substituted with a group selected from H, C1 to C5 alkyl, aryl, aralkyl, heteroaralkyl and arylsulfonyl. This latter embodiment represents compounds of the structure
wherein the nitrogen attached to R22 (not attached to the C═O) is also referred to herein as the second nitrogen of the piperazine and R22 is substituted with a group selected from H, C1 to C5 alkyl, aryl, aralkyl, heteroaralkyl and arylsulfonyl and wherein the latter groups, other than hydrogen, may themselves be substituted.
In additional preferred embodiments, NR13(CH2)nR14 is selected from N,N-dialkyl, N-alkyl-N-alkenyl, N-alkyl-N-alkylaminoalkyl and N-alkyl-N-alkoxyalkyl. Further preferred embodiments include compounds combining any or all of these preferred embodiments as structural limitations.
In any of the structures of the invention, R14 may be selected from any of H, C1 to C5 alkyl, C1 to C5 alkenyl, C1 to C5 alkoxy, cycloalkyl, OR15, SR15, or NR15R16 (wherein R15 and R16 are each independently selected from H and C1 to C5 alkyl); heterocycloalkyl having up to 3 heteroatoms selected from N or O and wherein when said heteroatom is N, it may be further substituted as may any carbon in said ring; phenyl or polyaromatic, heteroaryl with heteroatom N or O, aralkyl and alkylaryl; as well as F, Cl, Br, I, OH, CF3, NR15R16 (wherein R15 and R16 are each independently selected from H and C1 to C5 alkyl); wherein it may be substituted or unsubstituted, with substitutions selected from hydrogen, methyl, hydroxyl, sulfhydryl, alkoxy, thioalkoxy, alkyl, halogen, CN, CF3, NO2, cycloalkyl, heterocycloalkyl, aryl, COOR17, CONR18R19, NR18R19, NR18COR19, NR18SO2R19, NR17CONR18R19, wherein R17, R18, and R19 are independently as recited for R2 and wherein each said cycloalkyl, heterocycloalkyl, and aryl may be further substituted with a group selected from R2 as described elsewhere herein.
wherein
including all pharmaceutically acceptable salts, derivatives, prodrugs, metabolites, solvates, hydrates, and isomers thereof.
In a preferred embodiment of the compounds of Formula II, n=2. In other preferred embodiments, m=2. In yet other preferred embodiments, R9 is H, Cl or OMe. In still other preferred embodiments thereof, when NR13(CH2)nR14 is piperazine the ring N not attached to the C═O may be substituted with a group selected from H, C1 to C5 alkyl, aryl, aralkyl, heteroaralkyl and arylsulfonyl.
In additional preferred embodiments, NR13(CH2)nR14 is selected from N,N-dialkyl, N-alkyl-N-alkenyl, N-alkyl-N-alkylaminoalkyl and N-alkyl-N-alkoxyalkyl. Further preferred embodiments include compounds combining any or all of these preferred embodiments as structural limitations.
wherein
including all pharmaceutically acceptable salts, derivatives, prodrugs, metabolites, solvates, hydrates, and isomers thereof.
In a preferred embodiment of the compounds of Formula III, n=2. In other preferred embodiments, m=2. In yet other preferred embodiments, R9 is H, Cl or OMe. In still other preferred embodiments thereof, when NR13(CH2)nR14 is piperazine the ring N not attached to the C═O may be substituted with a group selected from H, C1 to C5 alkyl, aryl, aralkyl, heteroaralkyl and arylsulfonyl.
In additional preferred embodiments, NR13(CH2)nR14 is selected from N,N-dialkyl, N-alkyl-N-alkenyl, N-alkyl-N-alkylaminoalkyl and N-alkyl-N-alkoxyalkyl. Further preferred embodiments include compounds combining any or all of these preferred embodiments as structural limitations.
wherein
including all pharmaceutically acceptable salts, derivatives, prodrugs, metabolites, solvates, hydrates, and isomers thereof and as to all uses thereof but the invention is drawn specifically to compounds of Formula IV as such only when n is 1 and R3 is not H, Cl or OMe.
In a preferred embodiment of the compounds of Formula IV, n=2. In other preferred embodiments, m=2. In yet other preferred embodiments, R10 is H, Cl or OMe. In still other preferred embodiments thereof, when NR13(CH2)nR14 is piperazine the ring N not attached to the C═O may be substituted with a group selected from H, C1 to C5 alkyl, aryl, aralkyl, heteroaralkyl and arylsulfonyl.
In additional preferred embodiments, NR13(CH2)nR14 is selected from N,N-dialkyl, N-alkyl-N-alkenyl, N-alkyl-N-alkylaminoalkyl and N-alkyl-N-alkoxyalkyl. Further preferred embodiments include compounds combining any or all of these preferred embodiments as structural limitations.
The present invention also relates to compounds having the structure:
wherein
including all pharmaceutically acceptable salts, derivatives, prodrugs, metabolites, solvates, hydrates, and isomers thereof.
In a preferred embodiment of the compounds of Formula V, n=2. In other preferred embodiments, m=2. In yet other preferred embodiments, R10 is H, Cl or OMe. In still other preferred embodiments thereof, when NR13(CH2)nR14 is piperazine the ring N not attached to the C═O may be substituted with a group selected from H, C1 to C5 alkyl, aryl, aralkyl, heteroaralkyl and arylsulfonyl.
In additional preferred embodiments, NR13(CH2)nR14 is selected from N,N-dialkyl, N-alkyl-N-alkenyl, N-alkyl-N-alkylaminoalkyl and N-alkyl-N-alkoxyalkyl. Further preferred embodiments include compounds combining any or all of these preferred embodiments as structural limitations.
The present invention further relates to compounds of the structure
wherein
including all pharmaceutically acceptable salts, derivatives, prodrugs, metabolites, solvates, hydrates, and isomers thereof.
In a preferred embodiment of the compounds of Formula VI, n=2. In other preferred embodiments, m=2. In yet other preferred embodiments, R10 is H, Cl or OMe. In still other preferred embodiments thereof, when NR13(CH2)nR14 is piperazine the ring N not attached to the C═O may be substituted with a group selected from H, C1 to C5 alkyl, aryl, aralkyl, heteroaralkyl and arylsulfonyl.
In additional preferred embodiments, NR13(CH2)nR14 is selected from N,N-dialkyl, N-alkyl-N-alkenyl, N-alkyl-N-alkylaminoalkyl and N-alkyl-N-alkoxyalkyl. Further preferred embodiments include compounds combining any or all of these preferred embodiments as structural limitations.
In a highly preferred embodiment, the compounds of the invention are those with structures found in Table 1.
In a highly preferred embodiment, the compounds of the invention are those with structures found in Table 2.
In a highly preferred embodiment, the compounds of the invention are those with structures found in Table 3.
In a highly preferred embodiment, the compounds of the invention are those with structures found in Table 4A and 4B.
In another aspect, the present invention relates to compositions of any of the compounds of the invention, preferably wherein such compound is present in a pharmaceutically acceptable carrier and in a therapeutically effective amount. Such compositions will generally comprise an amount of such compound that is not toxic (i.e., an amount that is safe for therapeutic uses).
In accordance with the foregoing, the present invention is directed to use of the compounds of the invention as active ingredients for medicaments, in particular for medicaments useful for the treatment of tumors. The compounds of the invention will thus be present in pharmaceutical compositions containing compounds of formulas I to VI as active ingredients, in admixture with pharmaceutically acceptable vehicles and excipients, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol and ethanol, and the like, including carriers useful in forming sprays for nasal and other respiratory tract delivery or for delivery to the ophthalmic system. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. current edition). Use of such carriers is well known to those skilled in the art and will not be discussed further herein.
Also in accordance with the foregoing, the present invention relates to a method for preventing or treating a disease associated with a change in levels of expression of particular sets of genes in a mammal comprising administering to said mammal an effective amount of a compound of the invention.
Compounds according to the present invention will have the effect of reducing size and number of tumors, especially primary tumors, in a mammal, especially a human, in need of such treatment. A statistically significant change in the numbers of primary tumor or metastasizing cells will typically be at least about 10%, preferably 20%, 30%, 50%, 70%, 90%, or more.
In accordance with the present invention, the agents described herein may be combined with other treatments of the medical conditions described herein, such as other chemotherapies, radiation treatments, immunotherapy, surgical treatments, and the like. The compounds of the invention may also be administered in combination with such other agents as painkillers, diuretics, antidiuretics, antivirals, antibiotics, nutritional supplements, anemia therapeutics, blood clotting therapeutics, bone therapeutics, and psychiatric and psychological therapeutics.
Determination of the appropriate treatment dose is made by the clinician, e.g., using parameters or factors known in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects.
The phrase “effective amount” means an amount sufficient to effect a desired response, or to ameliorate a symptom or sign, e.g., of metastasis or primary tumor progression, size, or growth. Typical mammalian hosts will include mice, rats, cats, dogs, and primates, including humans. An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, route, and dose of administration and the severity of side affects. Preferably, the effect will result in a change in quantitation of at least about 10%, preferably at least 20%, 30%, 50%, 70%, or even 90% or more. When in combination, an effective amount is in ratio to a combination of components and the effect is not limited to individual components alone.
An effective amount of a therapeutic will modulate the symptoms typically by at least about 10%; usually by at least about 20%; preferably at least about 30%; or more preferably at least about 50%. Alternatively, modulation of migration will mean that the migration or trafficking of various cell types is affected. Such will result in, e.g., statistically significant and quantifiable changes in the numbers of cells being affected. This may be a decrease in the numbers of target cells being attracted within a time period or target area. Rate of primary tumor progression, size, or growth may also be monitored.
In another aspect, the present invention relates to a method for preventing or treating a disorder modulated by altered gene expression, wherein the disorder is selected from the group consisting of cancer, cardiovascular disorders, arthritis, osteoporosis, inflammation, periodontal disease and skin disorders, comprising administering to a mammal in need of such treatment or prevention a therapeutically effective amount of a compound of the invention.
In a preferred embodiment thereof, the disorder is cancer, more preferably colon cancer, most preferably adenocarcinoma, and the treatment prevents, arrests or reverts tumor growth, metastasis or both.
In a preferred embodiment, the present invention relates to a method of preventing, treating or ameliorating cancer or tumor metastasis in a mammal comprising administering to said mammal an effective a compound of the invention, preferably where said mammal is a human.
The compounds of the invention will commonly exert a therapeutic effect by modulation of one or more genes found in a cell, especially a mammalian cell, such as a cancer cell, preferably colon cancer and most preferably adenocarcinoma. Thus, a compound, or compounds, of the invention can be used to determine or demarcate a set of genes by determining modulation of such set of genes by one or more compounds of the invention. For example, where a set of genes is found to be up regulated in cancer cells versus otherwise normal cells, especially normal cells of the same tissue or organ as the cancer cells, a set of genes can be determined by their common property of being modulated (based on a change in expression of the genes, such as a change in rate or amount of RNA transcribed or the amount of polypeptide produced by said expression) by contacting such genes, or a cell containing such genes, with one or more of the compounds of the invention. The extent of such modulation may, of course, be related to the amount of said compound, or compounds, used in the contacting. Such modulation may include the increased expression of all the determined genes (i.e., the genes of the set), the decreased expression of all genes of the set, or the increase in expression of some of the genes of the set and decreased expression of others. Thus, a gene not modulated by the test compound (the compound used in contacting the genes or cell containing them) is not considered a member of the set.
Thus, the present invention relates to a gene set wherein expression of each member of said gene set is modulated as a result of contacting said gene set with a compound of the invention. In specific embodiments, expression of each member of said gene set is increased as a result of said contacting or is decreased as a result of said contacting. In another preferred embodiment, the gene set is present in a cell. Such a gene set will commonly be related to a specific disease process, such as a set of genes all of which are modulated by a compound of the invention wherein such compound has a specific therapeutic effect, such as being an anti-neoplastic agent.
In another aspect, the present invention relates to a method for identifying an agent that modulates the expression of a gene set of the invention, comprising:
(a) contacting, or otherwise using, a compound, such as a test compound, a test system, such as a source of genes or polynucleotides, for example, those found to be related to a given disease or disorder, or a set that is modulated by a given compound, or group of compounds, especially where these are found in a cell, so that the cell represents the test system, containing one or more polynucleotides corresponding to each of the members of the gene set of the invention under conditions wherein the members of said gene set are being expressed;
(b) determining a change in expression of each of said one or more polynucleotides of step (a) as a result of said treatment;
wherein said change in expression of step (b) indicates modulation of the members of said gene set by the test compound thereby identifying a test compound that modulates the expression of said gene set.
In one embodiment, the cell is a naturally derived cell that contains genes of a gene set or may be a recombinant cell engineered to comprise the genes or polynucleotides of the gene set. In an alternative embodiment, the test system may comprise the genes or polynucleotides in a cell-free system.
In a related aspect, the present invention provides a method for identifying a test compound that modulates the expression of a gene set, such as a gene set of the invention, comprising:
(a) contacting a test compound with one or more polynucleotides corresponding to each of the members of the gene set of the invention under conditions wherein the members of said gene set are being expressed;
(b) determining a change in expression of each of said one or more polynucleotides of step (a) as a result of said contacting;
wherein said change in expression of step (b) indicates modulation of the members of said gene set thereby identifying a test compound that modulates the expression of said gene set.
As used herein, “corresponding genes” or “corresponding polynucleotides” or “polynucleotides corresponding to genes” refers to polynucleotides and/or genes that encode an RNA that is at least 90% identical, preferably at least 95% identical, most preferably at least 98% identical, and especially identical, to an RNA encoded by one of the genes disclosed herein in Tables 8 and 9. Such genes will also encode the same polypeptide sequence, but may include differences in such amino acid sequences where such differences are limited to conservative amino acid substitutions, such as where the same overall three-dimensional structure, is maintained. A “corresponding gene” includes splice variants thereof.
The polynucleotides useful in the methods of the invention may be genomic in nature and thus represent the sequence of an actual gene, such as a human gene, or may be a cDNA sequence derived from a messenger RNA (mRNA) and thus represent contiguous exonic sequences derived from a corresponding genomic sequence, or they may be wholly synthetic in origin for purposes of practicing the processes of the invention. Because of the processing that may take place in transforming the initial RNA transcript into the final mRNA, the sequences disclosed herein may represent less than the full genomic sequence. They may also represent sequences derived from ribosomal and transfer RNAs. Consequently, the gene as present in the cell (and representing the genomic sequence) and the polynucleotide transcripts disclosed herein, including cDNA sequences, may be identical or may be such that the cDNAs contain less than the full genomic sequence. Such genes and cDNA sequences are still considered “corresponding sequences” (as defined elsewhere herein) because they both encode the same or related RNA sequences (i.e., related in the sense of being splice variants or RNAs at different stages of processing). Thus, by way of non-limiting example only, a gene that encodes an RNA transcript, which is then processed into a shorter mRNA, is deemed to encode both such RNAs and therefore encodes an RNA complementary to (using the usual Watson-Crick complementarity rules), or that would otherwise be encoded by, a cDNA (for example, a sequence as disclosed herein). Thus, the sequences disclosed herein correspond to genes contained in the cancerous cells (here, breast cancer) and are used to determine gene activity or expression because they represent the same sequence or are complementary to RNAs encoded by the gene. Such a gene also includes different alleles and splice variants that may occur in the cells used in the methods of the invention, such as where recombinant cells are used to assay for anti-neoplastic agents and such cells have been engineered to express a polynucleotide as disclosed herein, including cells that have been engineered to express such polynucleotides at a higher level than is found in non-engineered cancerous cells or where such recombinant cells express such polynucleotides only after having been engineered to do so. Such engineering includes genetic engineering, such as where one or more of the polynucleotides disclosed herein has been inserted into the genome of such cell or is present in a vector.
Such cells, especially mammalian cells, may also be engineered to express on their surfaces one or more of the polypeptides of the invention for testing with antibodies or other agents capable of masking such polypeptides and thereby removing the cancerous nature of the cell. Such engineering includes both genetic engineering, where the genetic complement of the cells is engineered to express the polypeptide, as well as non-genetic engineering, whereby the cell has been physically manipulated to incorporate a polypeptide of the invention in its plasma membrane, such as by direct insertion using chemical and/or other agents to achieve this result.
In a preferred embodiment of such method, the determined change in expression is a decrease in expression of said one or more polynucleotides or a decrease in said expression. In other preferred embodiments, the determined change in expression is a change in transcription of said one or more polynucleotides or a change in activity of a polypeptide, or expression product, encoded by said polynucleotide, including a change in the amount of said polypeptide synthesized, such as by a cell. The term “expression product” means that polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s).
In additional preferred embodiments, said one or more polynucleotides are present in a cell, preferably a cancer cell, more preferably a colon cancer cell, and most preferably where the colon cancer cell is an adenocarcinoma cancer cell. In another preferred embodiment of the invention, the cell is a recombinant cell engineered to contain said set of genes.
Such methods serve to identify other compounds that have like activity, including expected therapeutic activity, as the compounds of the invention and thus serve as the basis for large scale screening assays for therapeutic compounds. As a result, one or more compounds of the invention can be utilized to determine the presents of gene sets and subsets within the genome of a cell. Thus, the set of all genes modulated by a group of structurally related compounds of the invention can form a gene set while the different sets of genes regulated by each compound of a group will form a subset. By way of non-limiting example, where a structurally related group of 5 of the compounds of the invention (all having generally the structure of Formula I) modulate (by increasing or decreasing) expression of determined genes 1-20, this latter group of genes forms a gene set. Further examination then determines that genes 1-6 are modulated by compound A, genes 7-10 are modulated by compound B, genes 2-4 and 9-12 are modulated by compound C, genes 10-20 are modulated by compound D and the even numbered genes are modulated by compound E. Each of these groups of genes, such as the genes modulated by compound C, is considered a subset of the gene set of genes 1-20. In an analogous manner, the genes modulated by compound E can be themselves further subdivided into at least 2 subsets wherein one subset is made up of the genes whose expression is increased by compound E while the other subset is made up of genes whose expression is decreased by compound E, thus yielding subsets of subsets. It should be noted that within the context of the present invention, it is not necessary to identify subsets and that each so-called subset is, in its own right, a gene set as used in the invention. The identification of sets and subsets is thus a function of the extent that a user of the methods of the invention wishes to determine modulation of genes resulting from contacting of one or more compounds of the invention. Thus, the genes modulated by a single compound form a gene set and it is not necessary, in carrying out the methods of the invention, to compare different groups of genes for modulation by more than one compound but this may, of course, be done.
In accordance with the foregoing, the present invention relates to a set of genes comprising a plurality of subsets of genes wherein each subset of said plurality is a gene set identified by the methods of the invention. The present invention also relates to compounds identified as having activity using the methods of the invention, such as novel compounds not specifically described herein by structure but which have been identified by their ability to modulates one or more gene sets modulated by compounds of the invention.
In a preferred embodiment, the present invention encompasses the gene sets and subsets of the genes identified in Table 6 and/or in Table 7A or B. Using the compounds of the invention for treatment of disease, especially cancer, the present invention specifically contemplates use of a compound that modulates the expression of a set of, or subset of, genes of Table 7A or B.
The present invention also comprises methods for the preparation of compounds of the invention.
The compounds of the invention can be prepared using a variety of procedures known in the art. The starting materials used in preparing the compounds of the invention are known, made by known methods, or are commercially available. Particularly preferred syntheses are described in the following general reaction schemes.
Commercially available piperidine 1 is reacted with an ester 2 under standard Mitsunobu reaction conditions. The resulting ether 3 is subjected to acidic conditions under which the Boc protecting group is removed to produce amine 4. Substituent R2 is then introduced under standard reductive amination conditions using sodium triacetoxyborohydride. The intermediate ester is hydrolyzed under standard hydroxide-mediated conditions to produce acid 5. In the last step substituent R1 is introduced using EDAC mediated coupling reaction between acid 5 and an appropriate amine to produce compound 6.
Compounds for which no preparation is given can be made by methods known in the literature or are of common knowledge by skilled artisan.
The skilled artisan will recognize that some reactions are best carried out when another potentially reactive functionality on the molecule is masked or protected, thus avoiding any undesirable side reactions and/or increasing the yield of the reaction. Often protecting groups are used to accomplish such increased yields or to avoid the undesired reactions. Such reactions are well within the ability of the skilled artisan. Some examples are found in T. Greene, Protecting Groups in Organic Synthesis.
Step 1
To a solution of tert-butyl 4-hydroxypiperidine-1-carboxylate (5.64 g, 28 mmol) and methyl 3-chloro-4-hydroxybenzoate (3.73 g, 20 mmol) in anhydrous THF (200 ml) at room temperature is added triphenylphosphine (7.34 g, 28 mmol). DIAD (5.66 g, 28 mmol) is added dropwise over a one-hour period and the reaction is stirred at room temperature for 30 minutes. The reaction is quenched by addition of water (50 ml) and the mixture is extracted with ethyl acetate (3×100 ml). Combined organic extracts are washed with 0.1N HCl (80 ml), followed by water (80 ml) and brine, dried over sodium sulfate, filtered and concentrated under vacuum. The crude product is purified by flash column chromatography (80-20 hexane-ethyl acetate) to give the product as a colorless thick oil (6.52 g, 88% yield).
Step 2.
tert-Butyl 4-(2-chloro-4-(methoxycarbonyl)phenoxy)piperidine-1-carboxylate (2.75 g, 7.44 mmol) is dissolved in DCM (30 ml), TFA (4 mL) is added and the mixture is stirred at room temperature for 2 hrs. The mixture is concentrated under vacuum and partitioned between DCM (50 ml) and 0.5N NaOH (50 ml). The aqueous layer is extracted with DCM (50 ml) and the combined organic layers are dried over sodium sulfate, filtered, and concentrated under vacuum to give the product as a light yellow oil (1.85 g, 92% yield).
Step 3.
To a solution of 2,2-diphenylacetaldehyde (3.00 g, 14.9 mmol) and methyl 3-chloro-4-(piperidin-4-yloxy)benzoate (2.01 g, 7.44 mmol) in THF 30 ml) is added sodium triacetoxyborohydride (3.15 g, 14.9 mmol) and the mixture is stirred overnight under nitrogen at room temperature. The mixture is poured into EtOAc (50 ml) and extracted with 0.5N HCl (20 ml) and water (20 ml). The organic layer is then washed with brine, dried over sodium sulfate, filtered and concentrated under vacuum to give the crude product as an oil.
The crude product is dissolved in THF (30 ml) and MeOH (10 ml) and to the mixture is added aqueous NaOH (50% w/w solution, 2 mL). The mixture is stirred overnight at room temperature and ethyl acetate (100 ml) is added. The mixture is washed with 1N HCl (40 ml) followed by brine (2×30 ml) and the product is crystallized out of the organic phase. After filtration and drying the product is obtained as a white solid (2.0 g, 62% yield for both steps).
Step 4
To a solution of 3-chloro-4-(1-(2,2-diphenylethyl)piperidin-4-yloxy)benzoic acid (100 mg, 0.23 mmol), 2-(1-methylpyrrolidin-2-yl)ethanamine (29 mg, 0.23 mmol), and HOBT (102 mg, 0.75 mmol) in DCM (5 ml) is added EDAC (48 mg, 0.25 mmol) and the reaction is stirred overnight. The reaction mixture is diluted with DCM (20 ml) and extracted with 1N HCl (2×15 ml). The HCl washings were combined and made basic with aqueous NaOH, then extracted with DCM (3×10 ml). The combine organic phases are dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude material is purified by preparative HPLC to obtain the product as the free base, which is converted to the hydrochloride salt. The final product is obtained as a white solid (110 mg, 77% yield).
All other examples shown in tables below were prepared following the procedure described for Example 1 by using the appropriately substituted piperidine, benzoic acid ester and the corresponding R2 aldehyde and R1 amine.
In addition, it is to be appreciated that one optical isomer may have favorable properties over the other and thus the disclosure herein may include either optically active isomer if that isomer has advantageous physiological activity in accordance with the methods of the invention. Unless stated otherwise, the disclosure of an optically active isomer herein is intended to include all enantiomers or diastereomers of said compound so long as said structure has the activity described herein for the class of compounds of which said structure is a member.
This application claims priority of U.S. Provisional Application Ser. No. 60/774,972, filed 17 Feb. 2006, the disclosure of which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/04247 | 2/16/2007 | WO | 00 | 8/15/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60774972 | Feb 2006 | US |
