Patent Application
20050032794
The present invention relates to chemical agents affecting levels of gene expression in cellular systems, including cancer cells. In particular, the present invention relates to derivatives of quinone moiety, processes for their preparation, their use as antitumor drugs and pharmaceutical compositions containing them 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.
Our approach to screening small molecule compounds as potential anticancer drugs is based on the idea 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 quinones. Gene expression screening and subsequent cytotoxicity screening revealed that some of the compounds possess biological activity. Consequent, more detailed structure-activity relationship studies led to the discovery of compounds of formula I as new small molecule agents having antineoplastic activity.
In one aspect, the present invention relates to novel organic compounds, derivatives of quinone, 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 cancer such as colon and breast cancers.
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 and breast cancer cells, thereby producing an expression profile for said gene(s) that more resembles the expression profile found in normal cells. In another embodiment, the compounds disclosed herein are found to down regulate genes found to be up regulated in cancer cells, especially colon and breast 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 gene whose combined activity is related to a disease condition, such as cancer, especially colon and breast 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 and breast 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 and breast 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 4 carbon atoms. “Alkene” 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 4 carbon atoms. “Alkyne” 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, alkene and alkyne chains (referred to collectively as “hydrocarbon chains”) may be straight or branched and may be unsubstituted or substituted. Preferred branched alkyl, alkene and alkyne chains have one or two branches, preferably one branch. Preferred chains are alkyl. Alkyl, alkene and alkyne 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, alkene and alkyne 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, spirocycle, 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. p “Aryl” is an aromatic hydrocarbon ring. Aryl rings are monocyclic or fused bicyclic 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.
“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 have one or two branches, preferably one branch. Preferred heteroalkyl are saturated. Unsaturated heteroalkyl have one or more carbon-carbon double bonds and/or one or more carbon-carbon triple bonds. Preferred unsaturated heteroalkyls 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 are mono-, di-, or tri-substituted. Heteroalkyl may be substituted with lower alkyl, haloalkyl, halo, hydroxy, aryloxy, heteroaryloxy, acyloxy, carboxy, monocyclic aryl, heteroaryl, cycloalkyl, heterocycloalkyl, spirocycle, amino, acylamino, amido, keto, thioketo, cyano, or any combination thereof. Where a group is described, for example, as an alkyl derivative, such as “-ethylpyridine” the dash “-” indicate point of attachment of the substituent. Thus, “-ethylpyridine” means attachment of ethylpyridine via the ethyl portion of the group whereas “ethylpyridine-” means attachment via the pyridinyl ring.
“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:
While alkyl heteroalkyl, cycloalkyl, and heterocycloalkyl groups may be substituted with hydroxy, amino, and amido groups as stated above, the following are not envisioned in the invention:
Enols (OH attached to a carbon bearing a double bond).
Amino groups attached to a carbon bearing a double bond (except for vinylogous amides).
More than one hydroxy, amino, or amido attached to a single carbon (except where two nitrogen atoms are attached to a single carbon atom and all three atoms are member atoms within a heterocycloalkyl ring).
Hydroxy, amino, or amido attached to a carbon that also has a heteroatom attached to it.
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).
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 present invention relates generally to a compound having the structure:
wherein
In another preferred embodiment, R1, R2, R3, R4, and R5, are each selected from hydrogen, alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted polyaromatic, and substituted or unsubstituted heteroaromatic comprising one or more hetero atom(s) selected from N, O and S.
In another preferred embodiment, R1, R2, R3, R4, and R5 are each selected from substituted or unsubstituted aralkyl, substituted or unsubstituted cyclo or polycyclo hydrocarbon or mono or polyheterocycle (3-8 atoms per ring) with one to four hetero atoms selected from N, O and S.
In any of these preferred embodimenits, substitutions are selected from hydroxyl, sulfhydryl, lower alkoxy (1-6 carbon), lower thioalkoxy (1-6 carbon), lower alkyl (1-6 carbon), halogen, CN, CF3, NO2, COOR12, CONR12R13, NR12R13, NR12COR13, NR12SO2R13, and NR14CONR12R13, wherein R12, R13 and R14 are hydrogen, alkyl, heteroalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, or heterocycloalkyl. In a further preferred embodiment of the foregoing, R12 and R14 form a 4, 5, 6 or 7-member cyclic ring system.
In a further preferred embodiment, NR12R13 forms a substituted or unsubstituted mono or bicyclic ring comprising one to four heteroatoms selected from N, O and S.
In one preferred embodiment, R1, R4, R5, R6 and R7 are each selected from:
In a preferred embodiment of the compounds of Formula I, W and Z are each selected from C—R8, C—R11, and N, and X and Y are each selected from C—R9 and C—R9. In another preferred embodiment, X and Y are each selected from C—R9, C—R10 and N and wherein W and Z are each selected from C—R8 and C—R11. In another preferred embodiment, W is C—R8 or N, and X, Y and Z are each selected from C—R9, C—R10 and C—R11.
Where a position in a structure, such as W, X, Y or Z, or a substituent, such as an R group, as recited above, is described as selected from, it means that each of W, X, Y and Z, or R, can be selected from the indicated group of structures or atoms and each is selected independently of the others unless it is expressly stated herein to be otherwise. By “independent” is meant that the selection of one substituent does not limit the range of selection for another substituent, unless expressly stated as such. For example, where X and Y are selected from a range of atoms, such as N, O and S, then X and Y may be the same or different and the selection of one does not limit the range of the other. Thus, if X is nitrogen then Y can still be N, O or S.
Where a position, for example, in a ring, is described as being selected from “a bond” etc., this means that the position is not occupied by an atom. Thus, if in Formula I, X is a bond, then the ring with W, X, Y and Z is a 5 membered ring instead of a 6 membered ring.
In a preferred embodiment, NR4R5 and/or NR6R7 of Formula I form(s) a piperazine ring, preferably an N-acetylpiperazinyl group.
In a preferred embodiment, —NR4R5 and/or —NR6R7 of Formula I is a substituted or unsubstituted morpholinyl group. In a highly preferred embodiment thereof, R6 and R7 are both hydrogen. In a most preferred embodiment, R2 and R3 are both hydrogen and —NR4R5 forms an unsubstituted morpholinyl group.
In a preferred embodiment, NR4R5 and/or NR6R7 of Formula I is a piperidine ring, preferably a substituted piperidine ring, most preferably 4-hydroxypiperidine.
In a highly preferred embodiment of any of the structures of the present invention, R1, R6 and R7 of Formula I are each methyl.
In another preferred embodiment of the compounds of the invention, Z is C—R10, or N and W, Y and Z are each selected from C—R8, C—R9 and C—R11. In one embodiment of the latter, X is C—R10 or N and W, Y and Z are each selected from C—R8, C—R9 and C—R11. In a preferred embodiment of the latter Y is C—R10 or N and W, X, and Z are each selected from CH, C—R8, C—R9 and C—R11. In a most preferred embodiment thereof, W, X, Y and Z are each selected from CH, C—R8, C—R9, C—R10 and C—R11, most preferably where W X, Y and Z are each CH (thereby forming a phenyl ring).
In another preferred embodiment of the compounds of the invention, R2 and R3 are selected from hydrogen, lower alkyl (1-6 carbon) or aryl. In a further preferred embodiment of the compounds of the invention, R1 is selected from hydrogen, alkyl, cycloalkyl, unsubstituted or substituted phenyl, unsubstituted or substituted benzyl, -methylpyridine, -ethylpyridine, -methylindole, -ethylindole, alkoxyethyl-, hydroxyethyl-, N,N-diaikyl-ethyl, N,N-dialkyl-propyl, -methylpyrrole, -ethylpyrrole, -methylfuran, -ethylfuran, -alkylmorpholine, -alkylpiperizine, -alkypiperidine, and -alkylpyrrolidine and wherein R2 and R3 are selected from hydrogen, lower alkyl (1-6 carbons) and aryl.
In another preferred embodiment of the compounds of the invention, R4 and R5 are each selected from hydrogen, alkyl, cycloalkyl, unsubstituted or substituted phenyl, unsubstituted or substituted benzyl, -methylpyridine, -ethylpyridine, -methylindole, -ethylindole, alkoxyethyl-, hydroxyethyl-, N,N-dialkyl-ethyl-, N,N-dialkyl-propyl-, -methylpyrrole, -ethylpyrrole, -methylfuran, -ethylfuran, -alkyqmorpholine, -alkylpiperizine, -alkypiperidine, and -alkylpyrrolidine, and wherein R2 and R3 are selected from hydrogen, lower alkyl (1-6 carbon) and aryl.
In another preferred embodiment of the compounds of the invention, R6 and R7 are selected from alkyl, cycloalkyl, unsubstituted or substituted phenyl, unsubstituted or substituted benzyl, -methylpyridine, -ethylpyridine, -methylindole, -ethylindole, alkoxyethyl-, hydroxyethyl-, N,N-dialkyl-ethyl, N,N-dialkyl-propyl, -methylpyrrole, -ethylpyrrole, -methylfuran, -ethylfuran, -alkylmorpholine, -alkylpiperizine, -alkypiperidine, and -alkylpyrrolidine, and R2 and R3 are each selected from hydrogen, lower alkyl (1-6 carbons) and aryl.
In other preferred embodiments, R2 and R3 are selected from hydrogen, lower alkyl (1-6 carbon) and aryl, wherein R1, R4 and R5 are each selected from hydrogen, alkyl, cycloalkyl, unsubstituted or substituted phenyl, unsubstituted or substituted benzyl, -methylpyridine, -ethylpyridine, -methylindole, -ethylindole, alkoxyethyl-, hydroxyethyl-, N,N-dialkyl-ethyl-, N,N-dialkyl-propyl-, -methylpyrrole, -ethylpyrrole, -methylfuran, -ethylfuran, -alkylmorpholine, -alkylpiperizine, -alkypiperidine, and -alkylpyrrolidine, and wherein R2 and R3 are selected from hydrogen, lower alkyl (1-6 carbon) or aryl and wherein R6 and R7 are selected from alkyl, cycloalkyl, unsubstituted or substituted phenyl, unsubstituted or substituted benzyl, -methylpyridine, -ethylpyridine, -methylindole, -ethylindole, alkoxyethyl-, hydroxyethyl-, N,N-dialkyl-ethyl, N,N-dialkyl-propyl, -methylpyrrole, -ethylpyrrole, -methylfuran, -ethylfuran, -alkylmorpholine, -alkylpiperizine, -alkypiperidine, and -alkylpyrrolidine.
In another preferred embodiment of the compounds of the invention having Formula 1, R2 and R3 are each selected from hydrogen and alkyl, and wherein R4 and R6 are each selected from alkyl and
In another preferred embodimentof Formula I, R1 is alkyl while R2 and R3 are each selected from hydrogen and alkyl, and R4 and R6 are each selected from alkyl and
wherein n is 2, 3 or 4 and one or both of R5 and R7 is alkyl, preferably both, and in either case most preferably wherein the alkyl is methyl.
In another preferred embodiment of Formula 1, R2 and R3 are each selected from hydrogen and alkyl while R4 and R6 are each selected from alkyl and
In another preferred embodiment of Formula 1, R2 and R3 are each selected from hydrogen and alkyl, and R4 and R6 are each selected from alkyl and
In another preferred embodiment of Formula 1, R2 and R3 are each selected from hydrogen and alkyl and R4 and R6, are each selected from alkyl and
In another preferred embodiment of Formula 1, R2 and R3 are each be hydrogen or alkyl, R4 and R6 are each selected from alkyl and
In separate embodiments, the present invention encompasses compounds having a structure found in Table 1 including salts thereof, a compound having a structure of Table 2 including salts thereof, a compound having a structure of Table 3 including salts thereof, a compound having a structure of Table 4 including salts thereof, a compound having a structure of Table 5 including salts thereof, a compound having a structure of Table 6 including salts thereof, a compound having a structure of Table 7 including salts thereof, a compound having a structure of Table 8 including salts thereof, a compound having a structure of Table 9 including salts thereof, a compound having a structure of Table 11 including salts thereof, a compound having a structure of Table 12 including salts thereof, a compound having a structure of Table 13 including salts thereof, a compound having a structure of Table 14 including salts thereof, a compound having a structure of Table 15 including salts thereof, a compound having a structure of Table 16 including salts thereof, a compound having a structure of Table 17 including salts thereof, and a compound having a structure of Table 18 including salts thereof, and in each case most preferably pharmaceutically acceptable salts thereof. It is to be understood that each of the structures defined in each of these tables is considered to be a separate and preferred embodiment of the present invention.
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 formula I 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.
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.
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:
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:
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 Table 19. 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.
Because a polynucleotide or gene used in the methods of the invention “corresponds to” a gene present in one of the gene sets of the invention, such as genes identified in Table 19, such polynucleotide or gene encodes an RNA (processed or unprocessed, including naturally occurring splice variants and alleles) that is at least 90% identical, preferably at least 95% identical, most preferably at least 98% identical to, and especially identical to, an RNA that would be encoded by, or be complementary to, such as by hybridization with, a gene of Table 19, or genes of any gene set identified according to the invention. Polynucleotides encoding the same proteins as any of these genes, regardless of the percent identity of the sequences of such genes, and/or polynucleotides, are also specifically contemplated by any of the methods of the present invention. The polynucleotides used in the methods of the invention also include any open reading frames, as defined herein, present therein. As used herein, the term “open reading frame” (or ORF) means a series of triplets coding for amino acids without any termination codons and is a sequence (potentially) translatable into protein.
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 encqdes 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 asby 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 and breast cancer cell, and most preferably where the coloncancer 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 19.
The present invention comprises also processes for the preparation of compounds of formula I, and the relative key intermediates
Comp und Pr paration
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;
The dichloro compound 1 is either commercially available or can be synthesized using methods known in the literature.
The compound 1 is reacted with an amine in an appropriate solvent to provide the corresponding derivative 2. The compound 2 is then reacted with an appropriate 2-halo, 2-substituted acetyl halide to obtain the corresponding 3 derivatives. A reaction of crude or purified compound 3 with an amine gives compound 4. Compound 4 with or without isolation is treated with an amine in a suitable solvent at an appropriate temperature to afford compound 5.
In the same way, independent and selective modification of R1, R2, R3, R4, R5, R6, and R7 using methods known in the literature readily affords additional compounds of formula I. Thus, compounds for which no separate preparation is provided herein are made by methods known in the literature or are of common knowledge to the 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.
In addition, it is to be appreciated that one optical isomer may have favorable properties over the other and thus the disclosure of a racemic mixture within the present invention may also include either optically active isomer if such isomer has advantageous physiological activity in accordance with the methods of the invention.
2-Chloro-3-methylamino-[1,4]naphthoquinone
To a solution of 22.7 g (100 mmol, 1 equivalent) of 2,3-dichloro-[1,4]naphthopquinone in 350 ml of anhydrous THF was added 200 ml of 2.0M methyl amine in THF (200 mmol, 2 equivalents). To the mixture was added 34 ml of N, N-diisopropylethylamine (200 mmol, 2 equivalents) and it was shaken at room temperature for overnight (16-20 hours).
The red precipitates formed were filtered and washed with ether. The residue was again washed with water and ether. The solid was dried under vacuum. The filtrate was checked for the desired product, and then THF was evaporated. The residue was recrystallized with dichlorotnethane/ether. The titled compound was collected as a red solid (18 g, Yield 74%).
In a process analogous to Example A1 using appropriate starting materials, the corresponding compounds are prepared as follows:
2-Bromo-N-(3-chloro-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)-N-methyl-acetamide
To a solution of 8 g of 2-chloro-3-methylamino-[1,4]naphthoquinone (36 mmol) in 400 ml 1,4-dioxane was added 10 g of potassium carbonate (72 mmol). The mixture was heated until the starting material was completely dissolved. To the solution, 12.5 ml of bromoacetyl bromide (144 mmol) was added and refluxed for 1 hour. Inorganic materials were filtered and washed thoroughly with dichloromethane. The filtrate was evaporated and the residue was purified by flash silica gel column using 75:25-hexanes: ethyl acetate. The compound was collected as yellow oil. (10 g, Yield 80%).
In a process analogous to Example B1 using appropriate 2-chloro-3-substituted amino [1,4] naphthoquinone (Example A) and corresponding acid bromide following compounds are prepared.
2-Dimethylamino-N-(3-dimethylamino-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)-N-methyl-acetamide
To a solution of 2.5 g of 2-bromo-N-(3-chloro-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)-N-methyl-acetamide (7mmol, 1 equivalent) in 200 ml of ethyl acetate was added 28 ml of 2.0M dimethylamine solution in tetrahydrofuran (56 mmol, 8 equivalents). The amine solution was added in two portions stirring for 15 min after each addition. The solvent was then evaporated and then sample was purified on a silica gel column using initially ethyl acetate and then 10-20% methanol in ethyl acetate. The solvent was evaporated and the residue was dissolved in DMSO. It was then purified further on preparative LCMS using 0.1% NH4OH in water/acetonitrile as mobile phase. (592 mg, Yield 26%); H1 NMR (400 MHz, CDCl3) 2.97 (s, 6H), 3.08 (s, 3H), 3.20 (s, 6H), 3.64 (s, 2H), 7.62 (m, 2H), 7.95 (t, 2H).
Compound 2-119 (Table 1)
In a process analogous to Example 1 (Table 1) using appropriate chloro-bromo naphthoquinone (Example B) and the corresponding secondary amine, following compounds are prepared as shown in Table 1.
2-Chloro-N-(3-chloro-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)-N-methyl-acetamide
To a solution of 10 g of 2-chloro-3-methylamino-[1,4]naphthoquinone (45 mmol) in 250 mL of dioxane was added 172 mL of chloroacetyl chloride (48 equivalents). The reaction was heated at 85° C. for 16 hours. The solvent was evaporated and the material was purified on silica gel using DCM and hexanes as solvents. The pure fractions were combined and the solvent was evaporated. The product was collected as a yellow/brown solid. (12.1 g, Yield 90%).
In a process analogous to Example C1 using appropriate 2-chloro-3-substituted amino-[1,4 ]naphthoquinone (Example A) and corresponding acid chloride following compounds are prepared.
2-Chloro-N-(3-dimethylamino-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)-N-methyl-acetamide
To a solution of 19 g of 2-chloro-N-(3-chloro-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)-N-methyl-acetamide (63 mmol) in 200 mL of ethyl acetate was added slowly 22 mL of N, N-diisopropylethylamine (2 equivalents). 70 mL of 2.0M solution of dimethylamine in terahydrofuran (2.25 equivalents) was diluted with 100 mL of ethyl acetate. This amine solution was added slowly to the reaction mixture over one hour at room temperature. After stirring for an additional hour, the reaction was filtered and the solid material was washed with ethyl acetate. The filtrate was concentrated and purified using a normal phase column chromatography, and ethyl acetate and hexanes as solvents. The pure fractions were combined and the solvent was evaporated. The product was collected as a red solid. (10.1 g, Yield-52%). H1 NMR (400 MHz, CDCl3): 3.09 (s, 3H), 3.23 (s, 6H), 4.01 (q, 2H), 7.65-7.77 (m, 2H), 8.03 (d, 1H), 8.08 (d, 1H).
In a process analogous to Example D1 using appropriate dichloro naphthoquinone derivatives (Example C) and corresponding secondary amine, the following compounds are prepared.
2-Diethylamino-N-(3-dimethylamino-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)-N-methyl-acetamide
To a solution of 0.54 g of 2-chloro-N-(3-dimethylamino-1,4-dioxo-1,4-dihydro -naphthalen-2-yl)-N-methyl-acetamide (1.8 mmol) in 20 mL of ethyl acetate was added 2.2 mL of ethylamine (21.6 mmol, 12 equiv). The mixture was stirred at room temperature for two hours. The reaction mixture was then filtered and the solid was washed with ethyl acetate until all red material was dissolved. The red filtrate was concentrated and purified on a normal phase column chromatography using ethyl acetate. The pure fractions were combined and concentrated. The solid was then dissolved in 20 mL of DCM and 12 equiv of 1.0M HCl in diethyl ether was added to produce hydrochloride salt. Organic solvents were evaporated and the product was dissolved in 5.0 mL of HPLC grade water. This material was freeze dried to give 0.42 g of final product as its hydrochloride salt. (Yield 62%). H1 NMR (400 MHz, DMSO, D20) 1.14 (t, 6H), 2.97 (s, 3H), 3.08-3.0 (m, 10H), 3.80 (d, 1H), 4.02 (d, 1H), 7.7-7.9 (m, 2H), 7.89-8.0 (m, 2H).
Compounds 2-119 (Table 2)
In a process analogous to Example 2 using appropriate chloro naphthoquinone (Example D) and the corresponding secondary amine, following compounds are prepared as shown in Table 2.
Homo sapiens, clone IMAGE: 4826963, mRNA
Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 2344436.
Homo sapiens, clone IMAGE: 5285034, mRNA
Homo sapiens cDNA: FLJ22864 fis, clone KAT02164.
This application claims priority of U.S. Provisional Application Ser. No. 60/492,521, filed 5 Aug. 2003, and 60/523,477, filed 19 Nov. 2003, the disclosures of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60492521 | Aug 2003 | US | |
| 60523477 | Nov 2003 | US |
