Patent Application
20070015774
The present invention relates to compounds and compositions that are useful for treating cellular proliferative diseases, disorders associated with mutants of p53 activity, or in causing apoptosis of cancer cells. The compounds of the present invention are capable of restoring the biochemical and biological activity of mutant p53 and in causing apoptosis of cancer cells.
Cancer is a leading cause of death in the United States and throughout the world. Cancer cells are often characterized by constitutive proliferative signals, defects in cell cycle checkpoints, as well as defects in apoptotic pathways. There is a great need for the development of new chemotherapeutic drugs that can block cell proliferation and enhance apoptosis of tumor cells.
The p53 tumor suppressor protein belongs to a superfamily of transcription factors that includes its homologs p63 and p73. p53 is involved in a wide range of cellular activities that help ensure the stability of the genome, whereas p63 and p73 are involved in ectodermal morphogenesis, limb morphogenesis, neurogenesis, and homeostatic control and are not considered tumor suppressor genes (1). p53 is involved in DNA damage repair, cell cycle arrest, and apoptosis via transcriptional regulation of genes involved in these activities or by direct interaction with other proteins (2-4). Mutations that inactivate p53 are present in over 50% of all cancers and are indicative of aggressive cancers that are difficult to treat by chemotherapy or ionizing radiation (2, 5).
The majority of inactivating mutations reside in the central core DNA binding domain (DBD) of p53 (2, 5). These mutations can be divided into two main classes, DNA contact mutants, like R273H, where the mutation alters a residue involved in contact with DNA, and structural mutants, like R249S, which result in structural changes in the p53 core domain (−8). These mutations affect the function of p53 by distorting the structure and reducing the thermal stability of the protein (6-8). This can alter the ability of p53 to bind to various p53 response elements in a variety of genes, hampering its transcriptional regulation (9). In addition, these mutations may alter p53 structure, so that p53 can no longer induce apoptosis by binding to BcIXL, thereby inhibiting its anti-apoptotic function (10).
One potential therapeutic approach to cancer would be restoration of growth suppression activity to mutant p53. Several approaches have been tried, ranging from micro-injection of monoclonal antibody 421, C-terminal peptide of p53 and small molecules (11-16). Recently, small molecules and peptides, such as CP-31398, PRIMA1, and CDB3 peptide, have been shown to be effective in restoring p53 function (17-25). Both PRIMA1 and CDB3 have been shown to restore p53 DNA-binding activity in vitro (18-21), whereas the effects for CP-31398 have been shown primarily in cell-based assays (17, 22-25). Both CP-31398 and PRIMAL have been shown to reduce tumor size in animal models (17, 18). It is postulated that the two molecules perform similar tasks, but by different mechanisms. PRIMAL has been suggested to work more broadly to restore p53 DNA-binding activity, but the specific mechanism is not known (18). CP-31398, on the other hand, has been suggested to stabilize p53 as a protectant against thermal denaturation and maintain monoclonal antibody 1620 epitope conformation in newly synthesized p53 (17). Recently, CP-31398 has also been shown to stabilize wild type p53 in cells by inhibiting Mdm2-mediated ubiquitination and degradation (23). Reports from other studies suggest that CP-31398 interacts with DNA and not with p53 in vitro, and it is proposed to act as a DNA-damaging agent (26).
As indicated above, the p53 tumor suppressor protein is mutated in many human cancers and tumerogenicity can be inhibited by reintroduction of the wild type gene. Most of these mutations, which map to the central DBD, appear to cause conformational changes in the domain with loss of DNA binding and sequence specific transcriptional regulatory functions. Therefore, restoring transcriptional regulatory function to mutant p53 represents an attractive target to develop novel chemotherapeutics. The quinazoline derivatives of the present invention are anticancer agents that are capable of restoring the biochemical and biological activity of mutant p53 and in causing apoptosis of cancer cells.
International patent publication WO 2005/003100, published Jan. 13, 2005 refers to 4-arylaminoquinazolines and analogs as activators of caspases and inducers of apoptosis.
International patent publication WO 2004/014844, published Feb. 19, 2004, refers to substituted (2S)-(arylamino)-3-(biphenyl-4-yl)propionic acids as antagonists of factor IX for inhibiting the intrinsic pathway of blood coagulation.
In one embodiment, the present invention provides a compound represented by the structural Formula I:
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein
(i) X is OR4, SR5 or N(R6)2;
(ii) L is a linker selected from the group consisting of —N(R7)—, —N(R7)—(C═O)—, —N(R7)—(C═O)—N(R7)—, and —N(R7)—S(O)2—;
(iii) R1 and R2 are each independently selected from the group consisting of hydrogen and alkyl;
(iv) R3 is selected from the group of substituents consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclyl; wherein each of the aforesaid alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl and heterocyclyl substituents may optionally be independently substituted by one to four moieties independently selected from the group consisting of halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, alkyl-S—, alkyl-O-alkyl-O, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, fluorenyl, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—; wherein when each the aforesaid cycloalkyl, heterocyclyl, aryl and heteroaryl substituents contains two moieties on adjacent carbon atoms anywhere within said substituent, such moieties may optionally and independently in each occurrence, be taken together with the carbon atoms to which they are attached to form a five- to six-membered carbocyclic or heterocyclic ring; wherein each of the aforesaid moieties containing an aryl alternative may optionally be independently substituted by one or two radicals independently selected from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy;
wherein each of said aryl, cycloalkyl, heterocyclyl and heteroaryl moieties may optionally be independently substituted by one to two radicals selected independently from the group consisting of, methylenedioxy, alkyl-S—, aryl-S—, aryl-alkynyl-, alkyl-O—(C═O)-alkyl-O—, halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, fluorenyl, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—;
wherein when each of said aryl, heteroaryl, cycloalkyl, and heterocyclyl moieties contains two radicals on adjacent carbon atoms anywhere within said moiety, such radicals may optionally be taken together with the carbon atoms to which they are attached to form a five to six membered carbocyclic or heterocyclyl ring;
wherein each of the aforementioned radicals containing an aryl alternative may optionally be independently substituted by one or two radicals independently selected from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy;
(v) R4, R5, and each R6 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclyl, wherein each of the R4, R5, and R6 substituents alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl and heterocyclyl may optionally be independently substituted by one to four moieties independently selected from the group consisting of halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, H2N—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-H N—(C═O)-[(alkyl)—N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—NH—(C═O)—, alkyl-O—NH—(C═O)-alkyl-NH—(C═O)—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S—, alkyl-S(O)2—, aryl-S(O)2—, aryl-S—, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—; wherein when each of said cycloalkyl, heterocyclyl, heteroaryl, and aryl substituents contains two moieties on adjacent carbon atoms anywhere within said substituent, such moieties may optionally be taken together with the carbon atoms to which they are attached to form a five to six membered carbocyclic or heterocyclic ring, which carbocyclic or heterocyclyl ring may optionally be fused to an aryl ring;
wherein each of said aryl, cycloalkyl, heterocyclyl and heteroaryl moieties of said R4, R5, and R6 substituents may optionally be independently substituted by one to two radicals selected independently from the group consisting of, methylenedioxy, alkyl-S—, aryl-S—, aryl-alkynyl-, alkyl-O—(C═O)-alkyl-O—, halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, fluorenyl, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—; wherein each of said moieties containing an aryl alternative may optionally be substituted by one or two radicals independently selected from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy;
wherein when X is N(R6)2, the two R6 groups may optionally be taken together with the nitrogen atom to which they are shown attached to form a heterocyclyl or heteroaryl ring which heterocyclyl or heteroaryl ring may optionally be independently substituted with one to two substituents independently selected from the group consisting of halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, arylalkyl-, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, aryl-S(O)2—, aryl-S—, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—; wherein when each of said cycloalkyl, heterocyclyl, aryl, and heteroaryl substituents contains two moieties on adjacent carbon atoms anywhere within said substituent, such moieties may optionally and independently in each occurrence, be taken together with the carbon atoms to which they are attached to form a five to six membered carbocyclic or heterocyclic ring;
wherein each of the aforesaid R6 alkyl, alkenyl, aryl, arylalkyl-, cycloalkyl, heteroaryl, and heterocyclyl substituents may optionally be independently substituted with one to two moieties selected from the group consisting of halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, arylalkyl-, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, aryl-S(O)2—, aryl-S—, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—; wherein when each of said cycloalkyl, heterocyclycl, heteroaryl and aryl moieties contains two radicals on adjacent carbon atoms anywhere within said moiety, such radicals may optionally and independently in each occurrence, be taken together with the carbon atoms to which they are attached to form a five to six membered carbocyclic or heterocyclic ring; wherein each of the aforesaid moieties containing an aryl alternative may optionally be substituted by one or two radicals independently selected from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy; and
(vii) each R7 independently is selected from the group consisting of hydrogen, alkyl, and benzyl;
with the provisio that when L is —N(R8)— wherein R8 is unsubstituted alkyl, R3 is unsubstituted alkyl, and X is N(R6)2 wherein one R6 is unsubstitued alkyl, the other R6 is other than alkoxy substituted aryl.
Pharmaceutical formulations or compositions for the treatment of cellular proliferative diseases, for disorders associated with mutant p53 activity, for restoring biological or biochemical acitivity of mutant p53, and/or for causing apoptosis of cancer cells in a subject comprising administering a therapeutically effective amount of at least one of the inventive compounds and a pharmaceutically acceptable carrier to the subject also are provided.
Methods of treating cellular proliferative diseases, disorders associated with mutant p53 activity, for restoring biological or biochemical acitivity of mutant p53, and/or for causing apoptosis of cancer cells in a subject in need of such treatment an effective amount of at least one of the inventive compounds also are provided.
Processes for preparing the compound of formula I are also provided.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.”
In one embodiment, the present invention discloses compounds represented by structural Formula I or a pharmaceutically acceptable salt or ester thereof, wherein the various moieties are as described above.
In another embodiment, X in above Formula I is N(R6)2:
In another embodiment, in formula I, R1 and R2 are both hydrogen.
In another embodiment, in formula I, R3 is selected from the group of substituents consisting of alkyl and alkenyl;
wherein said alkyl and alkenyl substituents may optionally be independently substituted by one to four moieties selected independently from the group consisting of halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, alkyl-S—, alkyl-O-alkyl-O, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, fluorenyl, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—; wherein when each the aforesaid cycloalkyl, heterocyclyl, aryl and heteroaryl substituents contains two moieties on adjacent carbon atoms anywhere within said substituent, such moieties may optionally and independently in each occurrence, be taken together with the carbon atoms to which they are attached to form a five- to six-membered carbocyclic or heterocyclic ring; wherein each of the aforesaid moieties containing an aryl alternative may optionally be independently substituted by one or two radicals independently selected from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy;
wherein each of said aryl, cycloalkyl, heterocyclyl and heteroaryl moieties may optionally be independently substituted by one to two radicals selected independently from the group consisting of, methylenedioxy, alkyl-S—, aryl-S—, aryl-alkynyl-, alkyl-O—(C═O)-alkyl-O—, halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, fluorenyl, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—;
wherein when each of said aryl, heteroaryl, cycloalkyl, and heterocyclyl moieties contains two radicals on adjacent carbon atoms anywhere within said moiety, such radicals may optionally be taken together with the carbon atoms to which they are attached to form a five to six membered carbocyclic or heterocyclyl ring;
wherein each of the aforementioned radicals containing an aryl alternative may optionally be independently substituted by one or two radicals independently selected from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy.
In another embodiment, in formula I, the R3 alkyl and alkenyl substituents may optionally be independently substituted by one to two moieties selected independently from the group consisting of alkyl-S—, alkyl-O—(C═O)—, alkyl-O-alkyl-O—, fluorenyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, aryl-O—, aryl-S—, and aryl-S(O)2—;
wherein said aryl moiety may optionally be substituted by one or two radicals selected independently from the group consisting of halo, perhaloalkyl, perhaloalkoxy, cyano, —C(O)OH, hydroxy, alkyl, alkoxy, alkyl-S—, aryl, aryl-S—, aryl-alkynyl, alkyl-O—(C═O)—, and HO(O)C-alkyl-O—;
wherein said cycloalkyl moiety may optionally be substituted by an alkyl-O—(C═O)— radical;
wherein when said aryl and cycloalkyl moieties contain two radicals on adjacent carbon atoms, such radicals may optionally be taken together with the carbon atoms to which they are attached to form a five to six membered carbocyclic or heterocyclic ring;
wherein said heteroaryl moiety may optionally be substituted by one or two radicals selected independently from the group consisting of alkyl, heteroaryl, and aryl-S(O)2—;
wherein each of the aforementioned radicals containing an aryl alternative may optionally be substituted by one or two groups selected independently from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy.
In another embodiment, the cycloalkyl moiety of the R3 alkyl and alkenyl substituents is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopenyl, cyclohexyl and cycloheptyl, each of which may be optionally substituted.
In another embodiment, the heterocyclyl moiety of the R3 alkyl and alkenyl substituents is selected from the group consisting of piperidinyl, and dihydropyranyl, each of which may be optionally substituted.
In another embodiment, the heteroaryl moiety of the R3 alkyl and alkenyl substituents is selected from the group consisting of furanyl, thiophenyl, pyrrolyl,
each of which may be optionally substituted.
In another embodiment, in formula I, the aryl moiety of the R3 alkyl and alkenyl substituents, including aryl moiety containing two radicals on adjacent carbon atoms which are taken together with the carbon atoms to which said radicals are attached to form a five to six membered carbocyclic or heterocyclic ring, is selected from the group consisting of phenyl, naphthyl,
each of which may optionally be substituted.
In another embodiment, in formula I, R3 is selected from the group of substituents consisting of cycloalkyl, cycloalkenyl and heterocyclyl substituents,
wherein said cycloalkyl, cycloalkenyl and heterocyclyl substituents may optionally be independently substituted by one to four moieties selected independently from the group consisting of halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, alkyl-S—, alkyl-O-alkyl-O, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, fluorenyl, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—; wherein when each the aforesaid cycloalkyl, heterocyclyl, aryl and heteroaryl substituents contains two moieties on adjacent carbon atoms anywhere within said substituent, such moieties may optionally and independently in each occurrence, be taken together with the carbon atoms to which they are attached to form a five- to six-membered carbocyclic or heterocyclic ring; wherein each of the aforesaid moieties containing an aryl alternative may optionally be independently substituted by one or two radicals independently selected from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy;
wherein each of said aryl, cycloalkyl, heterocyclyl and heteroaryl moieties may optionally be independently substituted by one to two radicals selected independently from the group consisting of, methylenedioxy, alkyl-S—, aryl-S—, aryl-alkynyl-, alkyl-O—(C═O)-alkyl-O—, halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, fluorenyl, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—;
wherein when each of said aryl, heteroaryl, cycloalkyl, and heterocyclyl moieties contains two radicals on adjacent carbon atoms anywhere within said moiety, such radicals may optionally be taken together with the carbon atoms to which they are attached to form a five to six membered carbocyclic or heterocyclyl ring;
wherein each of the aforementioned radicals containing an aryl alternative may optionally be independently substituted by one or two radicals independently selected from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy.
In another embodiment, in formula I, the R3 cycloalkyl, cycloalkenyl and heterocyclyl substituents may optionally be independently substituted by one to four moieties selected independently from the group consisting of cyano, alkyl, aryl, alkyl-(C═O)—, aryl-(C═O)—, perhaloalkyl and perhaloalkoxy; wherein when said cycloalkyl, cycloalkenyl and heterocyclyl substituents contain two moieties on adjacent carbon atoms anywhere within said substituents, such moieties may optionally and independently in each occurrence, be taken together with the carbon atoms to which they are attached to form a five to six membered carbocyclic or heterocyclic ring; wherein when said cycloalkyl, cycloalkenyl and heterocyclyl substituents contain two moieties on the same carbon, such moieties may optionally be taken together with the carbon atom to which they are attached to form a five to six membered carbocyclic or heterocyclic ring.
In another embodiment, in formula I, the R3 cycloalkyl substituent, including including cycloalkyl substituent containing two moieties on adjacent carbon atoms which are taken together with the carbon atoms to which said moieties are attached to form a five to six membered carbocyclic or heterocyclic ring, and including cycloalkyl substituent containing moieties on the same carbon which are taken together with the carbon atom to which said moieties are attached to form a five to six membered carbocyclic or heterocyclic ring, is selected from the group consisting of multicyclic ring system, cyclopropyl, cyclobutyl, cyclopenyl, cyclohexyl, cycloheptyl, polycycloalkyl,
each of which may optionally be substituted.
In another embodiment, in formula I, the R3 heterocyclyl substituent, including heterocyclyl substituent containing two moieties on adjacent carbon atoms which are taken together with the carbon atoms to which said moieties are attached to form a five to six membered carbocyclic or heterocyclic ring, is selected from the group consisting of tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, piperidinyl,
each of which may be optionally substituted.
In another embodiment, in formula I, R3 is selected from the group of substituents consisting of aryl and heteroaryl;
wherein said aryl and heteroaryl substituents may optionally be independently substituted by one to four moieties selected independently from the group consisting of halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, alkyl-S—, alkyl-O-alkyl-O, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, fluorenyl, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—; wherein when each the aforesaid cycloalkyl, heterocyclyl, aryl and heteroaryl substituents contains two moieties on adjacent carbon atoms anywhere within said substituent, such moieties may optionally and independently in each occurrence, be taken together with the carbon atoms to which they are attached to form a five- to six-membered carbocyclic or heterocyclic ring; wherein each of the aforesaid moieties containing an aryl alternative may optionally be independently substituted by one or two radicals independently selected from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy;
wherein each of said aryl, cycloalkyl, heterocyclyl and heteroaryl moieties may optionally be independently substituted by one to two radicals selected independently from the group consisting of, methylenedioxy, alkyl-S—, aryl-S—, aryl-alkynyl-, alkyl-O—(C═O)-alkyl-O—, halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, fluorenyl, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—;
wherein when each of said aryl, heteroaryl, cycloalkyl, and heterocyclyl moieties contains two radicals on adjacent carbon atoms anywhere within said moiety, such radicals may optionally be taken together with the carbon atoms to which they are attached to form a five to six membered carbocyclic or heterocyclyl ring;
wherein each of the aforementioned radicals containing an aryl alternative may optionally be independently substituted by one or two radicals independently selected from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy.
In another embodiment, in formula I, the R3 aryl and heteroaryl substituents may optionally be independently substituted by one to four moieties selected independently from the group consisting of cyano, halo, alkyl, alkoxy, aryloxy, alkyl-S—, alkyl-(C═O)—NH—, alkyl-O—(C═O)—, perfluoroalkyl, perfluoroalkoxy, aryl, cycloalkyl, aralkyl-, and cyanoalkyl; wherein each of said moieties containing an aryl alternative may optionally be substituted by one or two radicals independently selected from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy;
wherein when said aryl and heteroaryl substituents contain two moieties on adjacent carbon atoms anywhere within said substituents, such moieties may optionally and independently in each occurrence, be taken together with the carbon atoms to which they are attached to form a five to six membered carbocyclic or heterocyclic ring.
In another embodiment, in formula I, the R3 aryl substituent, including aryl substituent containing two moieties on adjacent carbon atoms which are taken together with the carbon atoms to which said moieties are attached to form a five to six membered carbocyclic or heterocyclic ring, is selected from the group consisting of phenyl, naphthyl,
each of which may be optionally substituted.
In another embodiment, in formula I, the R3 heteroaryl substituent, including heteroaryl substituent containing two moieties on adjacent carbon atoms which are taken together with the carbon atoms to which said moieties are attached to form a five to six membered carbocyclic or heterocyclic ring, is selected from the group consisting of pyridinyl, furanyl, thiophenyl, pyrrolyl,
each of which may be optionally substituted.
In another embodiment, in formula I, X is N(R6)2; one R6 is selected from the group of substituents consisting of hydrogen or alkyl, and the other R6 is selected from the group of substituents consisting of alkyl, cycloalkyl, heterocyclyl, heteroaryl and aryl; wherein each of the aforesaid other R6 alkyl, cycloalkyl, heterocyclyl, heteroaryl and aryl substituents may optionally be independently substituted by one to four moieties selected from the group consisting of halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, arylalkyl-, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)-N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, aryl-S(O)2—, aryl-S—, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—; wherein when each of said cycloalkyl, heterocyclyl, aryl, and heteroaryl substituents contains two moieties on adjacent carbon atoms, such moieties may optionally be taken together with the carbon atoms to which they are attached to form a five to six membered carbocyclic or heterocyclic ring;
wherein each of said aryl, cycloalkyl, heterocyclyl and heteroaryl moieties may optionally be independently substituted by one to two radicals selected independently from the group consisting of, methylenedioxy, alkyl-S—, aryl-S—, aryl-alkynyl-, alkyl-O—(C═O)-alkyl-O—, halo, alkyl, alkenyl, alkynyl, perhaloalkyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, formyl, —C≡N, alkyl-(C═O)—, aryl-(C═O)—, HO—(C═O)—, alkyl-O—(C═O)—, alkyl-NH—(C═O)—, (alkyl)2—N—(C═O)—, aryl-NH—(C═O)—, aryl-[(alkyl)-N]—(C═O)—, —NO2, amino, alkylamino, (alkyl)2-amino, alkyl-(C═O)—NH—, alkyl-(C═O)-[(alkyl)-N]—, aryl-(C═O)—NH—, aryl-(C═O)-[(alkyl)-N]—, H2N—(C═O)—, H2N—(C═O)—NH—, alkyl-HN—(C═O)—NH—, (alkyl)2—N—(C═O)—NH—, alkyl-HN—(C═O)-[(alkyl)-N]—, (alkyl)2—N—(C═O)-[(alkyl)-N]—, aryl-HN—(C═O)—NH—, (aryl)2-N—(C═O)—NH—, aryl-HN—(C═O)-[(alkyl)-N]—, (aryl)2-N—(C═O)-[(alkyl)-N]—, alkyl-O—(C═O)—NH—, alkyl-O—(C═O)-[(alkyl)—N]—, aryl-O—(C═O)—NH—, aryl-O—(C═O)-[(alkyl)-N]—, alkyl-S(O)2NH—, aryl-S(O)2NH—, alkyl-S(O)2—, fluorenyl, hydroxy, alkoxy, perhaloalkoxy, aryloxy, alkyl-(C═O)—O—, aryl-(C═O)—O—, H2N—(C═O)—O—, alkyl-HN—(C═O)—O—, (alkyl)2—N—(C═O)—O—, aryl-HN—(C═O)—O— and (aryl)2-N—(C═O)—O—; wherein each of said moieties containing an aryl alternative may optionally be substituted by one or two radicals independently selected from the group consisting of alkyl, halo, alkoxy, cyano, perhaloalkyl and perhaloalkoxy.
In another embodiment, in formula I, X is N(R6)2; one R6 is hydrogen, and the other R6 is alkyl substituted by one or two moieties selected from the group consisting of alkyl-(C═O)—, H2N—(C═O)—, and (alkyl)2-amino. In another embodiment, in formula I, X is N(R6)2; wherein said N(R6)2 is selected from the group consisting of
In another embodiment, the compound of formula I is selected from the group consisting of
or a pharmaceutically acceptable salt, solvate or ester thereof.
In the above table of compounds, the compound # corresponds to the particular example # set forth in the “EXAMPLES” section below where the preparation of such compound is shown. Where a compound has two numbers separated by a dash (−), the first number represents the example # where the preparation of the compound is shown, and the second number designates an arbitrary number for the particular compound. Thus compound #32-1 indicates that this a compound whose preparation is shown in Example 11. Similarly #32-2 indicates a different compound whose preparation is also shown in Example 11. The notation “PE” before a compound # refers to “Preparative Example”. Thus compound “PE-20” refers to a compound whose preparation is shown in “Preparative Example 20” in the “EXAMPLES” section.
In another preferred embodiment, the compound of formula I is selected from the group consisting of
or a pharmaceutically acceptable salt, solvate or ester thereof.
In another more preferred embodiment, the compound of formula I is selected from the group consisting of
or a pharmaceutically acceptable salt, solvate or ester thereof.
In another embodiment, the present invention provides a compound of the formula
or a pharmaceutically acceptable salt, solvate or ester thereof.
the formula
or a pharmaceutically acceptable salt, solvate or ester thereof.
In other embodiments, the present invention provides processes for producing such compounds, pharmaceutical formulations or compositions comprising one or more of such compounds, and methods of treating or preventing one or more conditions or diseases associated with p53 mutant activity such as those discussed in detail below.
As used above, and throughout the specification, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
“Subject” includes both mammals and non-mammalian animals.
“Mammal” includes humans and other mammalian animals.
The term “substituted” means that one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. By “stable compound” or “stable structure” is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
The term “optionally substituted” means optional substitution with the specified groups, radicals or moieties. It should be noted that any atom with unsatisfied valences in the text, schemes, examples and tables herein is assumed to have the hydrogen atom(s) to satisfy the valences.
The terms “substituent”, “moiety” and “radical” have specific and distinct meanings as used herein and represent a hierarchy in the use of such terms. The hierarchy used generally is “substituent”→“moiety”→“radical”, starting out with “substituent” and ending with “radical” while describing the branching out of various groups. Thus, for example, a specific R group will be described as being selected from a group of specified substituents. The substituents will then be described as having certain “moieties”, and those moieties will be described as having certain “radicals”. Thus an “alkyl substituent” as used herein is differentiated from an “alkyl moiety” which in turn is differentiated from an “alkyl radical”. Such use of terminology is generally adhered to consistently throughout the specification for preservation of proper antecedent basis.
The term “aryl alternative” refers to a certain “moiety” or “radical” wherein said “moiety” or “radical” contains an aryl group as part a larger group. For example, in the phrase, “ . . . substituted by one to four moieties selected from alkyl, alkoxy, perfluoroalkyl, aryloxy, aryl-O—(C═O)—NH, aryl-S(O)2NH, and aryl-HN—(C═O)—O—, wherein each of the aforesaid moieties containing an aryl alternative may optionally be independently substituted by one or two radicals selected from the group consisting of halo, alkyl and cyano”, the term “aforesaid moieties containing an aryl alternative” refers to the aryloxy, aryl-O—(C═O)—NH, aryl-S(O)2NH, and aryl-HN—(C═O)—O— moieties, and it is the aryl group within these aryloxy, aryl-O—(C═O)—NH, aryl-S(O)2NH, and aryl-HN—(C═O)—O— moieties that may be substituted with the halo, alkyl and cyano radicals.
The following definitions apply regardless of whether a term is used by itself or in combination with other terms, unless otherwise indicated. Therefore, the definition of “alkyl” applies to “alkyl” as well as the “alkyl” portions of “hydroxyalkyl”, “haloalkyl”, “alkoxy”, etc.
As used herein, the term “alkyl” means an aliphatic hydrocarbon group which may be straight or branched and comprising about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups contain about 1 to about 12 carbon atoms in the chain. More preferred alkyl groups contain about 1 to about 6 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkyl chain. “Lower alkyl” means a group having about 1 to about 6 carbon atoms in the chain which may be straight or branched. The alkyl group may be substituted with one or more substituents independently selected from the group consisting of halo, alkyl, aryl, cycloalkyl, cyano, hydroxy, alkoxy, amino, —NH(alkyl), —NH(cycloalkyl), —N(alkyl)2, carboxy, —C(O)O-alkyl and —S(alkyl), wherein said alkyl, cycloalkyl and aryl are unsubstituted. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, heptyl, nonyl, decyl, fluoromethyl, trifluoromethyl and cyclopropylmethyl. Whenever applicable, the term “alkyl” also includes a divalent alkyl, i.e., an “alkylene” group, obtained by removal of a hydrogen atom from an alkyl group. Examples of alkylene groups include methylene (—CH2—), ethylene (—CH2CH2—), propylene (—C3H6—) and the like including where applicable both straight chain and branched structures.
“Alkenyl” means an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched and comprising about 2 to about 15 carbon atoms in the chain. Preferred alkenyl groups have about 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 6 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkenyl chain. “Lower alkenyl” means about 2 to about 6 carbon atoms in the chain which may be straight or branched. The alkenyl group may be substituted with one or more substituents independently selected from the group consisting of halo, alkyl, aryl, cycloalkyl, cyano, alkoxy and —S(alkyl), wherein said alkyl, cycloalkyl and aryl are unsubstituted. Non-limiting examples of suitable alkenyl groups include ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl, octenyl and decenyl.
“Alkynyl” means an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and which may be straight or branched and comprising about 2 to about 15 carbon atoms in the chain. Preferred alkynyl groups have about 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkynyl chain. “Lower alkynyl” means about 2 to about 6 carbon atoms in the chain which may be straight or branched. Non-limiting examples of suitable alkynyl groups include ethynyl, propynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, and decynyl. The alkynyl group may be substituted with one or more substituents being independently selected from the group consisting of alkyl, aryl and cycloalkyl, wherein said alkyl, cycloalkyl and aryl are unsubstituted.
“Alkoxy” means an alkyl-O— group in which the alkyl group is as previously described. Useful alkoxy groups can comprise 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms. Non-limiting examples of suitable alkoxy groups include methoxy, ethoxy and isopropoxy. The alkyl group of the alkoxy is linked to an adjacent moiety through the ether oxygen.
The term “perhaloalkyl” means, unless otherwise stated, alkyl substituted with (2m′+1) halogen atoms, where m′ is the total number of carbon atoms in the alkyl group. For example, the term “perhaloalkyl” includes trifluoromethyl, pentachloroethyl, 1,1,1-trifluoro-2-bromo-2-chloroethyl, and the like.
The term “perhaloalkoxy” means, unless otherwise stated, alkyloxy (i.e., alkoxy) substituted with (2m′+1) halogen atoms, where m′ is the total number of carbon atoms in the alkoxy group. For example, the term “perhaloalkoxy” includes trifluoromethoxy, pentachloroethoxy, 1,1,1-trifluoro-2-bromo-2-chloroethoxy, and the like.
“Aryl” means an aromatic monocyclic or multicyclic ring system comprising about 5 to about 14 carbon atoms, preferably about 6 to about 10 carbon atoms. The aryl group can be substituted with one or more “ring system substituents” which may be the same or different, and are as defined herein. Non-limiting examples of suitable aryl groups include phenyl and naphthyl. Also included within the scope of the term “aryl”, as used herein, is a group in which an aromatic hydrocarbon ring is fused to one or more non-aromatic carbocyclic or heteroatom-containing rings, such as in an indanyl, phenanthridinyl or tetrahydronaphthyl, where the radical or point of attachment is on the aromatic hydrocarbon ring.
“Aralkyl” or “arylalkyl” means an alkyl group substituted with an aryl group in which the aryl and alkyl are as previously described. Preferred aralkyls comprise a lower alkyl group. Non-limiting examples of suitable aralkyl groups include benzyl, phenethyl and naphthlenylmethyl. The aralkyl is linked to an adjacent moiety through the alkylene group.
“Cycloalkyl” means a non-aromatic mono- or multicyclic hydrocarbon ring system comprising about 3 to about 12 carbon atoms, preferably about 5 to about 10 carbon atoms. Preferred cycloalkyl rings contain about 5 to about 7 ring atoms. The cycloalkyl can be substituted with one or more “ring system substituents” which may be the same or different, and are as defined below. Non-limiting examples of suitable monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. Non-limiting examples of suitable multicyclic cycloalkyls include 1-decalinyl, norbornyl, adamantyl and the like. A cycloalkyl may be fully saturated or may contain one or more units of unsaturation but is not aromatic. The term “cycloalkyl” also includes hydrocarbon rings that are fused to one or more aromatic rings where the radical or point of attachment is on the non-aromatic ring.
“Halo” or halogen refers to fluorine, chlorine, bromine or iodine radicals. Preferred are fluorine, chlorine and bromine
“Heteroaryl” means a monocyclic or multicyclic aromatic ring system of about 5 to about 14 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the atoms in the ring system is/are atoms other than carbon, for example nitrogen, oxygen or sulfur. Preferred heteroaryls contain about 5 to about 6 ring atoms. The “heteroaryl” can be optionally substituted with one or more “ring system substituents” which may be the same or different, and are as defined herein. The prefix aza, oxa or thia before the heteroaryl root name means that at least a nitrogen, oxygen or sulfur atom respectively, is present as a ring atom. A nitrogen atom of a heteroaryl can be oxidized to form the corresponding N-oxide. All regioisomers are contemplated, e.g., 2-pyridyl, 3-pyridyl and 4-pyridyl. Examples of useful 6-membered heteroaryl groups include pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl and the like and the N-oxides thereof. Examples of useful 5-membered heteroaryl rings include furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl and isoxazolyl. Useful bicyclic groups are benzo-fused ring systems derived from the heteroaryl groups named above, e.g., quinolyl, phthalazinyl, quinazolinyl, benzofuranyl, benzothienyl and indolyl. Also included within the scope of the term “heteroaryl” is a group in which a heteroaromatic ring is fused to one or more aromatic or non-aromatic rings where the radical or point of attachment is on the heteroaromatic ring. The term “heteroaryl” also refers to partially saturated heteroaryl moieties such as, for example, tetrahydroisoquinolyl, tetrahydroquinolyl and the like.
“Heteroarylalkyl” or “heteroaralkyl” means an alkyl group substituted with a heteroaryl group in which the heteroaryl and alkyl are as previously described. Preferred heteroaralkyls contain a lower alkyl group. Non-limiting examples of suitable heteroaralkyl groups include pyridylmethyl, 2-(furan-3-yl)ethyl and quinolin-3-ylmethyl. The bond to the parent moiety is through the alkyl. “Heteroarylalkoxy” means a heteroaryl-alkyl-O— group in which the heteroaryl and alkyl are as previously described.
“Heterocyclyl” means a non-aromatic monocyclic or multicyclic ring system comprising about 3 to about 12 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the atoms in the ring system is an element other than carbon, for example nitrogen, oxygen or sulfur, or combinations thereof. Preferred heterocyclyls contain about 5 to about 6 ring atoms. The prefix aza, oxa or thia before the heterocyclyl root name means that at least a nitrogen, oxygen or sulfur atom respectively is present as a ring atom. The heterocyclyl can be optionally substituted with one or more “ring system substituents” which may be the same or different, and are as defined herein. The nitrogen or sulfur atom of the heterocyclyl can be optionally oxidized to the corresponding N-oxide, S-oxide or S-dioxide. Non-limiting examples of suitable monocyclic heterocyclyl rings include piperidyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,3-dioxolanyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, lactam, lactone, and the like. A heterocyclic ring may be fully saturated or may contain one or more units of unsaturation but is not aromatic.
“Heterocyclylalkyl” means an alkyl group substituted with a heterocyclyl group in which the heterocyclyl and alkyl groups are as previously described. Preferred heterocyclylalkyls contain a lower alkyl group. The bond to the parent moiety is through the alkyl.
“Ring system substituent” means a substituent attached to an aromatic or non-aromatic ring system that, for example, replaces an available hydrogen on the ring system. Ring system substituents may be the same or different, each being independently selected from the group consisting of aryl, heteroaryl, aralkyl, alkylaryl, aralkenyl, heteroaralkyl, alkylheteroaryl, heteroaralkenyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, acyl, aroyl, halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkyl, cycloalkenyl, heterocyclyl, heterocyclenyl, Y1Y2N—, Y1Y2N-alkyl-, Y1Y2NC(O)— and Y1Y2NSO2—, wherein Y1 and Y2 may be the same or different and are independently selected from the group consisting of hydrogen, alkyl, aryl, and aralkyl. “Ring system substituent” may also mean a single moiety which simultaneously replaces two available hydrogens on two adjacent carbon atoms (one H on each carbon) on a ring system. Examples of such moiety are methylene dioxy, ethylenedioxy, —C(CH3)2— and the like which form moieties such as, for example:
“Hydroxyalkyl” means a HO-alkyl-group in which alkyl is as previously defined. Preferred hydroxyalkyls contain lower alkyl. Non-limiting examples of suitable hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.
“Alkylamino” means an —NH2 or —NH3+ group in which one or more of the hydrogen atoms on the nitrogen is replaced by an alkyl group as defined above.
“Haloalkyl” means a halo-alkyl-group in which alkyl is as previously defined. Preferred haloalkyls contain lower alkyl.
“Alkoxyalkyl” means an alkoxy-alkyl group in which alkyl is as previously defined. Preferred alkoxyalkyls contain lower alkyl.
Also included in the scope of this invention are oxidized forms of the heteroatoms (e.g., nitrogen and sulfur) that are present in the compounds of this invention. Such oxidized forms include N(O) [N+—O−], S(O) and S(O)2.
The term “isolated” or “in isolated form” for a compound refers to the physical state of said compound after being isolated from a synthetic process or natural source or combination thereof. The term “purified” or “in purified form” for a compound refers to the physical state of said compound after being obtained from a purification process or processes described herein or well known to the skilled artisan, in sufficient purity to be characterizable by standard analytical techniques described herein or well known to the skilled artisan.
When a functional group in a compound is termed “protected”, this means that the group is in modified form to preclude undesired side reactions at the protected site when the compound is subjected to a reaction. Suitable protecting groups will be recognized by those with ordinary skill in the art as well as by reference to standard textbooks such as, for example, T. W. Greene et al, Protective Groups in organic Synthesis (1991), Wiley, New York.
As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
Isomers of the compounds of Formula I (where they exist), including enantiomers, stereoisomers, rotamers, tautomers and racemates are also contemplated as being part of this invention. The invention includes d and I isomers in both pure form and in admixture, including racemic mixtures. Isomers can be prepared using conventional techniques, either by reacting optically pure or optically enriched starting materials or by separating isomers of a compound of the Formula I. Isomers may also include geometric isomers, e.g., when a double bond is present. Polymorphous forms of the compounds of Formula I, whether crystalline or amorphous, also are contemplated as being part of this invention. The (+) isomers of the present compounds are preferred compounds of the present invention.
Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by a 13C— or 14C-enriched carbon are also within the scope of this invention.
It will be apparent to one skilled in the art that certain compounds of this invention may exist in alternative tautomeric forms. All such tautomeric forms of the present compounds are within the scope of the invention. Unless otherwise indicated, the representation of either tautomer is meant to include the other. For example, both isomers (1) and (2) are contemplated:
wherein R′ is H or C16 unsubstituted alkyl.
Prodrugs and solvates of the compounds of the invention are also contemplated herein. The term “prodrug”, as employed herein, denotes a compound that is a drug precursor which, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield a compound of formula I or a salt, ester and/or solvate thereof (e.g., a prodrug on being brought to the physiological pH or through enzyme action is converted to the desired drug form). A discussion of prodrugs is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems (1987) Volume 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, (1987) Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press, both of which are incorporated herein by reference thereto.
“Solvate” means a physical association of a compound of this invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like. “Hydrate” is a solvate wherein the solvent molecule is H2O.
One or more compounds of the invention may also exist as, or optionally converted to, a solvate. Preparation of solvates is generally known. Thus, for example, M. Caira et al, J. Pharmaceutical Sci., 93(3), 601-611 (2004) describe the preparation of the solvates of the antifungal fluconazole in ethyl acetate as well as from water. Similar preparations of solvates, hemisolvate, hydrates and the like are described by E. C. van Tonder et al, AAPS Pharm Sci Tech., 5(1), article 12 (2004); and A. L. Bingham et al, Chem. Commun., 603-604 (2001). A typical, non-limiting, process involves dissolving a compound in desired amounts of the desired solvent (organic or water or mixtures thereof) at a higher than ambient temperature, and cooling the solution at a rate sufficient to form crystals which are then isolated by standard methods. Analytical techniques such as, for example I. R. spectroscopy, show the presence of the solvent (or water) in the crystals as a solvate (or hydrate).
“Effective amount” or “therapeutically effective amount” is meant to describe an amount of a compound or a composition of the present invention effective in inhibiting mitotic kinesins, in particular KSP kinesin activity, and thus producing the desired therapeutic, ameliorative, inhibitory or preventative effect in a suitable subject.
The compounds of formula I form salts which are also within the scope of this invention. Reference to a compound of formula I herein is understood to include reference to salts, esters and solvates thereof, unless otherwise indicated. The term “salt(s)”, as employed herein, denotes acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. In addition, when a compound of formula I contains both a basic moiety, such as, but not limited to a pyridine or imidazole, and an acidic moiety, such as, but not limited to a carboxylic acid, zwitterions (“inner salts”) may be formed and are included within the term “salt(s)” as used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful. Salts of the compounds of the formula I may be formed, for example, by reacting a compound of formula I with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization. Acids (and bases) which are generally considered suitable for the formation of pharmaceutically useful salts from basic (or acidic) pharmaceutical compounds are discussed, for example, by S. Berge et al, Journal of Pharmaceutical Sciences (1977) 66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33 201-217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; in The Orange Book (Food & Drug Administration, Washington, D.C. on their website); and P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (2002) Int'l. Union of Pure and Applied Chemistry, pp. 330-331. These disclosures are incorporated herein by reference thereto.
Exemplary acid addition salts include acetates, adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiod ides, 2-hydroxyethanesulfonates, lactates, maleates, methanesulfonates, methyl sulfates, 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pamoates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates, sulfonates (such as those mentioned herein), tartarates, thiocyanates, toluenesulfonates (also known as tosylates,) undecanoates, and the like.
Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, aluminum salts, zinc salts, salts with organic bases (for example, organic amines) such as benzathines, diethylamine, dicyclohexylamines, hydrabamines (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, piperazine, phenylcyclohexylamine, choline, tromethamine, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups may be quarternized with agents such as lower alkyl halides (e.g. methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g. decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g. benzyl and phenethyl bromides), and others.
All such acid salts and base salts are intended to be pharmaceutically acceptable salts within the scope of the invention. All acid and base salts, as well as esters and solvates, are considered equivalent to the free forms of the corresponding compounds for purposes of the invention.
Pharmaceutically acceptable esters of the present compounds include the following groups: (1) carboxylic acid esters obtained by esterification of the hydroxy groups, in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, acetyl, n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted with, for example, halogen, C1-4alkyl, or C1-4alkoxy or amino); (2) sulfonate esters, such as alkyl- or aralkylsulfonyl (for example, methanesulfonyl); (3) amino acid esters (for example, L-valyl or L-isoleucyl); (4) phosphonate esters and (5) mono-, di- or triphosphate esters. The phosphate esters may be further esterified by, for example, a C1-20 alcohol or reactive derivative thereof, or by a 2,3-di (C6-24)acyl glycerol.
In such esters, unless otherwise specified, any alkyl moiety present preferably contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Any cycloalkyl moiety present in such esters preferably contains from 3 to 6 carbon atoms. Any aryl moiety present in such esters preferably comprises a phenyl group.
Generally, the compounds of Formula I can be prepared by a variety of methods as disclosed in the examples hereinbelow.
One embodiment of the present invention provides a process for preparing the compound of formula I as set forth above, but wherein L is a linker selected from the group consisting of —N(R7)—(C═O)—, —N(R7)—S(O)2—, and —N(R7)—(C═O)—N(H)—, wherein R7 is selected from the group consisting of alkyl and benzyl comprises reacting a compound of formula II
with R3C(O)Cl, R3S(O)2Cl, or R3NCO wherein each R3 independently and X, R1, R2, and R7 in formula II are as set forth in formula I above; wherein reacting the compound of formula II R3C(O)Cl, R3S(O)2Cl, or R3NCO produces the compound with formula I wherein L is respectively —N(R7)—(C═O)—, —N(R7)—S(O)2—, and —N(R7)—(C═O)—N(H)—.
Another embodiment of the present invention refers to the method of preparing the compound of formula I, wherein X is N(R6)2.
Another embodiment of the present invention relates to a process for preparing a compound of formula I, wherein L is a linker that is —N(R7)—; wherein R7 is selected from the group consisting of alkyl and benzyl; comprising reacting a compound of formula III
with R4OH, R5SH and HN(R6)2; wherein Y in formula III is a halogen; R1, R2, R3 in formula III are as set forth in formula I; L in formula III is is —N(R7)—; R4, R5, and R6 in R4OH, R5SH and HN(R6)2 respectively are set forth in formula I; and wherein reacting the compound of formula III with R4OH, R5SH and HN(R6)2 produces the compound with formula I wherein X is respectively OR4, SR5 and N(R6)2.
Another embodiment of the present invention refers to the above mentioned process for preparing a compound of formula I utilizing the compound of formula III wherein X is N(R6)2, and wherein the compound of formula III is reacted with HN(R6)2.
Another embodiment of the present invention refers to the above mentioned process for preparing a compound of formula I utilizing the compound of formula II, wherein X is N(R6)2, wherein R1 and R2 are both hydrogen in formula I and formula III.
Another embodiment of the present invention refers to the above mentioned process for preparing a compound of formula I utilizing the compound of formula II, wherein the compound of formula III is made by reacting a compound of compound of formula IV
with P(O)Y3, wherein Y is a halogen.
Another embodiment of the present invention refers to the above mentioned process for preparing a compound of formula III utilizing the compound of formula IV and P(O)Y3 whrein Y is chlorine.
The compounds of the invention can be used to treat cellular proliferation diseases. Such disease states which can be treated by the compounds, compositions and methods provided herein include, but are not limited to, cancer (further discussed below), hyperplasia, cardiac hypertrophy, autoimmune diseases, fungal disorders, arthritis, graft rejection, inflammatory bowel disease, immune disorders, inflammation, cellular proliferation induced after medical procedures, including, but not limited to, surgery, angioplasty, and the like. Treatment includes inhibiting cellular proliferation. It is appreciated that in some cases the cells may not be in a hyper- or hypoproliferation state (abnormal state) and still require treatment. For example, during wound healing, the cells may be proliferating “normally”, but proliferation enhancement may be desired. Thus, in one embodiment, the invention herein includes application to cells or subjects afflicted or subject to impending affliction with any one of these disorders or states.
The compounds, compositions and methods provided herein are particularly useful for the treatment of cancer including solid tumors such as skin, breast, brain, colon, gall bladder, thyroid, cervical carcinomas, testicular carcinomas, etc. More particularly, cancers that may be treated by the compounds, compositions and methods of the invention include, but are not limited to:
Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma;
Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;
Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma);
Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma);
Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma;
Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors;
Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma);
Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma);
Hematologic: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, acute and chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma), B-cell lymphoma, T-cell lymphoma, hairy cell lymphoma, Burkett's lymphoma, promyelocytic leukemia;
Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis;
Adrenal glands: neuroblastoma; and
Other tumors: including xenoderoma pigmentosum, keratoctanthoma and thyroid follicular cancer.
As used herein, treatment of cancer includes treatment of cancerous cells, including cells afflicted by any one of the above-identified conditions.
The compounds of the present invention may also be useful in the chemoprevention of cancer. Chemoprevention is defined as inhibiting the development of invasive cancer by either blocking the initiating mutagenic event or by blocking the progression of pre-malignant cells that have already suffered an insult or inhibiting tumor relapse.
The compounds of the present invention may also be useful in inhibiting tumor angiogenesis and metastasis.
The compounds of the present invention may also be useful as antifungal agents, by modulating the activity of the fungal members of the bimC kinesin subgroup, as is described in U.S. Pat. No. 6,284,480.
The present compounds are also useful in combination with one or more other known therapeutic agents and anti-cancer agents. Combinations of the present compounds with other anti-cancer or chemotherapeutic agents are within the scope of the invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V. T. Devita and S. Hellman (editors), 6th edition (Feb. 15, 2001), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved. Such anti-cancer agents include, but are not limited to, the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, apoptosis inducing agents and agents that interfere with cell cycle checkpoints. The present compounds are also useful when co-administered with radiation therapy.
The phrase “estrogen receptor modulators” refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate, 4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-ydrazone, aid SH646.
The phrase “androgen receptor modulators” refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism. Examples of androgen receptor modulators include finasteride and other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.
The phrase “retinoid receptor modulators” refers to compounds which interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, a difluoromethylornithine, ILX23-7553, trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.
The phrase “cytotoxic/cytostatic agents” refer to compounds which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell mycosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, inhibitors of kinases involved in mitotic progression, antimetabolites; biological response modifiers; hormonal/anti-hormonal therapeutic agents, haematopoietic growth factors, monoclonal antibody targeted therapeutic agents, monoclonal antibody therapeutics, topoisomerase inhibitors, proteasome inhibitors and ubiquitin ligase inhibitors.
Examples of cytotoxic agents include, but are not limited to, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide (TEMODART from Schering-Plough Corporation, Kenilworth, N.J.), cyclophosphamide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, doxorubicin, irofulven, dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum(II)]tetrachloride, diarizidinylspermine, arsenic trioxide, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston, 3′-deansino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755, 4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunombicin (see WO 00/50032), methoxtrexate, gemcitabine, and mixture thereof.
An example of a hypoxia activatable compound is tirapazamine.
Examples of proteasome inhibitors include, but are not limited to, lactacystin and bortezomib.
Examples of microtubule inhibitors/microtubule-stabilising agents include paclitaxel, vindesine sulfate, 3′,4′-didehydro-4′-deoxy-8′-norvincaleu koblastine, docetaxel, rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881, BMS184476, vinflunine, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide, TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and 6,288,237) and BMS188797.
Some examples of topoisomerase inhibitors are topotecan, hycaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin, 9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H) propanamine, 1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione, lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350, BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2′-dimethylamino-2′-deoxy-etoposide, GL331, N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide, asulacrine, (5a, 5aB, 8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one, 2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium, 6,9-bis[(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione, 5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one, N-[1-[2-(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide, N-(2-(dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one, dimesna, and camptostar.
Other useful anti-cancer agents that can be used in combination with the present compounds include thymidilate synthase inhibitors, such as 5-fluorouracil.
In one embodiment, inhibitors of mitotic kinesins include, but are not limited to, inhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E, inhibitors of MCAK, inhibitors of Kif14, inhibitors of Mphosphl and inhibitors of Rab6-KIFL.
The phrase “inhibitors of kinases involved in mitotic progression” include, but are not limited to, inhibitors of aurora kinase, inhibitors of Polo-like kinases (PLK) (in particular inhibitors of PLK-1), inhibitors of bub-1 and inhibitors of bub-R1.
The phrase “antiproliferative agents” includes antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001, and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, neizarabine, 2′-deoxy-2′-methylidenecytidine, 2′-fluoromethylene-2′-deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine, aplidine, ecteinascidin, troxacitabine, 4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-flurouracil, alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase, 2′-cyano-2′-deoxy-N-4-palmitoyl-1-B-D-arabino furanosyl cytosine and 3-aminopyridine-2-carboxaldehyde thiosemicarbazone.
Examples of monoclonal antibody targeted therapeutic agents include those therapeutic agents which have cytotoxic agents or radioisotopes attached to a cancer cell specific or target cell specific monoclonal antibody. Examples include Bexxar.
Examples of monoclonal antibody therapeutics useful for treating cancer include Erbitux (Cetuximab).
The phrase “HMG-CoA reductase inhibitors” refers to inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase. Examples of HMG-CoA reductase inhibitors that may be used include but are not limited to lovastatin (MEVACOR®; see U.S. Pat. Nos. 4,231,938, 4,294,926 and 4,319,039), simvastatin(ZOCOR®; see U.S. Pat. Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; see U.S. Pat. Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®; see U.S. Pat. Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896) and atorvastatin (LIPITOR®; see U.S. Pat. Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952). The structural formulas of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are described at page 87 of M. Yalpani, “Cholesterol Lowering Drugs”, Chemistry & Industry, pp. 85-89 (5 Feb. 1996) and U.S. Pat. Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefore the use of such salts, esters, open acid and lactone forms is included in the scope of this invention.
The phrase “prenyl-protein transferase inhibitor” refers to a compound which inhibits any one or any combination of the prenyl-protein transferase enzymes, including farnesyl-protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and geranylgeranyl-protein transferase type-II (GGPTase-II, also called Rab GGPTase).
Examples of prenyl-protein transferase inhibitors can be found in the following publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO 95/32987, U.S. Pat. Nos. 5,420,245, 5,523,430, 5,532,359, 5,510,510, 5,589,485, 5,602,098, European Patent Publ. 0 618 221, European Patent Publ. 0 675 112, European Patent Publ. 0 604181, European Patent Publ. 0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO 95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO 96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO, 97/30053, WO 97/44350, WO 98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of a prenyl-protein transferase inhibitor on angiogenesis see European of Cancer, Vol. 35, No. 9, pp. 1394-1401(1999).
Examples of farnesyl protein transferase inhibitors include SARASAR™ (4-[2-[4-[(11R)-3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl-]-1-piperidinyl]-2-oxoehtyl]-1-piperidinecarboxamide from Schering-Plough Corporation, Kenilworth, N.J.), tipifarnib (Zarnestra® or R115777 from Janssen Pharmaceuticals), L778,123 (a farnesyl protein transferase inhibitor from Merck & Company, Whitehouse Station, N.J.), BMS 214662 (a farnesyl protein transferase inhibitor from Bristol-Myers Squibb Pharmaceuticals, Princeton, N.J.).
The phrase “angiogenesis inhibitors” refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, interferon-α (for example Intron and Peg-Intron), interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal anti-inflammatories (NSAIDs) like aspirin and ibuprofen as well as selective cyclooxygenase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch. Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin. Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p. 107 (1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodies to VEGF (see, Nature Biotechnology, Vol. 17, pp. 963-968 (October 1999); Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).
Other therapeutic agents that modulate or inhibit angiogenesis and may also be used in combination with the compounds of the instant invention include agents that modulate or inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examples of such agents that modulate or inhibit the coagulation and fibrinolysis pathways include, but are not limited to, heparin (see Thromb. Haemost. 80:10-23 (1998)), low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354 (2001)). Examples of TAFIa inhibitors have been described in PCT Publication WO 03/013,526.
The phrase “agents that interfere with cell cycle checkpoints” refers to compounds that inhibit protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing the cancer cell to DNA damaging agents. Such agents include inhibitors of ATR, ATM, the Chk1 and Chk2 kinases and cdk and cdc kinase inhibitors and are specifically exemplified by 7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.
The phrase “inhibitors of cell proliferation and survival signaling pathway” refers to agents that inhibit cell surface receptors and signal transduction cascades downstream of those surface receptors. Such agents include inhibitors of EGFR (for example gefitinib and erlotinib), antibodies to EGFR (for example C225), inhibitors of ERB-2 (for example trastuzumab), inhibitors of IGFR, inhibitors of cytokine receptors, inhibitors of MET, inhibitors of P13K (for example LY294002), serine/threonine kinases (including but not limited to inhibitors of Akt such as described in WO 02/083064, WO 02/083139, WO 02/083140 and WO 02/083138), inhibitors of Raf kinase (for example BAY43-9006), inhibitors of MEEK (for example CI-1040 and PD-098059), inhibitors of mTOR (for example Wyeth CCI-779), and inhibitors of C-abl kinase (for example GLEEVEC™, Novartis Pharmaceuticals). Such agents include small molecule inhibitor compounds and antibody antagonists.
The phrase “apoptosis inducing agents” includes activators of TNF receptor family members (including the TRAIL receptors).
In one embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a combination of at least one comound of formula I or a pharmaceutically acceptable salt, solvate or ester thereof and temozolomide.
In another embodiment, the present invention provides a method of treating a proliferative disease in a subject comprising administering to said subject in need of such treatment a therapeutically effective amount of a combination of of at least one comound of formula I or a pharmaceutically acceptable salt, solvate or ester thereof and temozolomide.
In another embodiment, the present invention provides a process for potentiating the growth activity suppression activity of temolozamide in cancer cells comprising administering to said cells therapeutically effective amount of a combination of at least one compound of formula I or a pharmaceutically acceptable salt, solvate or ester thereof and temozolomide.
In another embodiment, the cancer cells useful in the above process for potentiating the growth activity suppression activity of temolozolamide is selected from the group consisting of pancreatic and glioma cells.
The invention also encompasses combinations with NSAID's which are selective COX-2 inhibitors. For purposes of this specification NSAID's which are selective inhibitors of COX-2 are defined as those which possess a specificity for inhibiting COX-2 over COX-1 of at least 100 fold as measured by the ratio of IC50 for COX-2 over IC50 for COX-1 evaluated by cell or microsomal assays. Inhibitors of COX-2 that are particularly useful in the instant method of treatment are: 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and 5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5 pyridinyl)pyridine; or a pharmaceutically acceptable salt thereof.
Compounds that have been described as specific inhibitors of COX-2 and are therefore useful in the present invention include, but are not limited to, parecoxib, CELIEBREX® and BEXTRA® or a pharmaceutically acceptable salt thereof.
Other examples of angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, ranpirnase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide, CM101, squalamine, combretastatin, RP14610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalene disulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416).
As used above, “integrin blockers” refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the αvβ3 integrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the αvβ5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the αvβ3 integrin and the αvβ5 integrin, and to compounds which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells. The term also refers to antagonists of the αvβ6, αvβ8, α1β1, α2β1, α5β1, α6β1 and α6β4 integrins. The term also refers to antagonists of any combination of αvβ3, αvβ5, αvβ6, αvβ8, α1β1, α2β1, α5βα6β1 and α6β4 integrins.
Some examples of tyrosine kinase inhibitors include N-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine, BIBX1382, 2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one, SH268, genistein, ST1571, CEP2563, 4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethane sulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, ST1571A, N4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.
Combinations with compounds other than anti-cancer compounds are also encompassed in the instant methods. For example, combinations of the present compounds with PPAR-γ (i.e., PPAR-gamma) agonists and PPAR-δ (i.e., PPAR-delta) agonists are useful in the treatment of certain malingnancies. PPAR-γ and PPAR-δ are the nuclear peroxisome proliferator-activated receptors γ and δ. The expression of PPAR-γ on endothelial cells and its involvement in angiogenesis has been reported in the literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J. Biol. Chem. 1999;274:9116-9121; Invest. Ophthalmol Vis. Sci. 2000; 41:2309-2317). More recently, PPAR-γ agonists have been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit the development of retinal neovascularization in mice (Arch. Ophthamol. 2001; 119:709-717). Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are not limited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, G1262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionic acid, and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid.
In one embodiment, useful anti-cancer (also known as anti-neoplastic) agents that can be used in combination with the present compounds include, but are not limited, to Uracil mustard, Chlormethine, Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, Dacarbazine, Floxuridine, Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, oxaliplatin, leucovirin, oxaliplatin (ELOXATIN™ from Sanofi-Synthelabo Pharmaeuticals, France), Pentostatine, Vinblastine, Vincristine, Vindesine, Bleomycin, Dactinomycin, Daunorubicin, Doxonubicin, Epirubicin, Idarubicin, Mithramycin, Deoxycoformycin, Mitomycin-C, L-Asparaginase, Teniposide 17α-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate, Methylprednisolone, Methyltestosterone, Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, goserelin, Cisplatin, Carboplatin, Hydroxyurea, Amsacrine, Procarbazine, Mitotane, Mitoxantrone, Levamisole, Navelbene, Anastrazole, Letrazole, Capecitabine, Reloxafine, Droloxafine, Hexamethylmelamine, doxorubicin (adriamycin), cyclophosphamide (cytoxan), gemcitabine, interferons, pegylated interferons, Erbitux and mixtures thereof.
Another embodiment of the present invention is the use of the present compounds in combination with gene therapy for the treatment of cancer. For an overview of genetic strategies to treating cancer, see Hall et al (Am J Hum Genet 61:785-789,1997) and Kufe et al (Cancer Medicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapy can be used to deliver any tumor suppressing gene. Examples of such genes include, but are not limited to, p53, which can be delivered via recombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134, for example), a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of a uPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in Mice,” Gene Therapy, August 1998; 5(8):1105-13), and interferon gamma (J Immunol 2000; 164:217-222).
The present compounds can also be administered in combination with one or more inhibitor of inherent multidrug resistance (MDR), in particular MDR associated with high levels of expression of transporter proteins. Such MDR inhibitors include inhibitors of p-glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar).
The present compounds can also be employed in conjunction with one or more anti-emetic agents to treat nausea or emesis, including acute, delayed, late-phase, and anticipatory emesis, which may result from the use of a compound of the present invention, alone or with radiation therapy. For the prevention or treatment of emesis, a compound of the present invention may be used in conjunction with one or more other anti-emetic agents, especially neurokinin-1 receptor antagonists, 5HT3 receptor, antagonists, such as ondansetron, granisetron, tropisetron, and zatisetron, GABAB receptor agonists, such as baclofen, a corticosteroid such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or those as described in U.S. Pat. Nos. 2,789,118, 2,990,401, 3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, an antidopaminergic, such as the phenothiazines (for example prochlorperazine, fluphenazine, thioridazine and mesoridazine), metoclopramide or dronabinol. In one embodiment, an anti-emesis agent selected from a neurokinin-1 receptor antagonist, a 5HT3 receptor antagonist and a corticosteroid is administered as an adjuvant for the treatment or prevention of emesis that may result upon administration of the present compounds.
Examples of neurokinin-1 receptor antagonists that can be used in conjunction with the present compounds are described in U.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270, 5,494,926, 5,496,833, 5,637,699, and 5,719,147, content of which are incorporated herein by reference. In an embodiment, the neurokinin-1 receptor antagonist for use in conjunction with the compounds of the present invention is selected from: 2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine, or a pharmaceutically acceptable salt thereof, which is described in U.S. Pat. No. 5,719,147.
A compound of the present invention may also be administered with one or more immunologic-enhancing drug, such as for example, levamisole, isoprinosine and Zadaxin.
Thus, the present invention encompasses the use of the present compounds (for example, for treating or preventing cellular proliferative diseases) in combination with a second compound selected from: an estrogen receptor modulator, an androgen receptor modulator, retinoid receptor modulator, a cytotoxic/cytostatic agent, an antiproliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, a PPAR-δ agonist, an inhibitor of inherent multidrug resistance, an anti-emetic agent, an immunologic-enhancing drug, an inhibitor of cell proliferation and survival signaling, an agent that interfers with a cell cycle checkpoint, and an apoptosis inducing agent.
In one embodiment, the present invention empassesses the composition and use of the present compounds in combination with a second compound selected from: a cytostatic agent, a cytotoxic agent, taxanes, a topoisomerase II inhibitor, a topoisomerase I inhibitor, a tubulin interacting agent, hormonal agent, a thymidilate synthase inhibitors, anti-metabolites, an alkylating agent, a farnesyl protein transferase inhibitor, a signal transduction inhibitor, an EGFR kinase inhibitor, an antibody to EGFR, a C-abl kinase inhibitor, hormonal therapy combinations, and aromatase combinations.
The term “treating cancer” or “treatment of cancer” refers to administration to a mammal afflicted with a cancerous condition and refers to an effect that alleviates the cancerous condition by killing the cancerous cells, but also to an effect that results in the inhibition of growth and/or metastasis of the cancer.
In one embodiment, the angiogenesis inhibitor to be used as the second compound is selected from a tyrosine kinase inhibitor, an inhibitor of epidermal-derived growth factor, an inhibitor of fibroblast-derived growth factor, an inhibitor of platelet derived growth factor, an MW (matrix metalloprotease) inhibitor, an integrin blocker, interferon-α, interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole, combretastatin A-4, squalamine, 6-(O-chloroacetylcarbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, or an antibody to VEGF. In an embodiment, the estrogen receptor modulator is tamoxifen or raloxifene.
Also included in the present invention is a method of treating cancer comprising administering a therapeutically effective amount of at least one compound of Formula I in combination with radiation therapy and at least one compound selected from: an estrogen receptor modulator, an androgen receptor modulator, retinoid receptor modulator, a cytotoxic/cytostatic agent, an antiproliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, a PPAR-δ agonist, an inhibitor of inherent multidrug resistance, an anti-emetic agent, an immunologic-enhancing drag, an inhibitor of cell proliferation and survival signaling, an agent that interfers with a cell cycle checkpoint, and an apoptosis inducing agent.
Yet another embodiment of the invention is a method of treating cancer comprising administering a therapeutically effective amount of at least one compound of Formula I in combination with paclitaxel or trastuzumab.
The present invention also includes a pharmaceutical composition useful for treating or preventing cellular proliferation diseases (such as cancer, hyperplasia, cardiac hypertrophy, autoimmune diseases, fungal disorders, arthritis, graft rejection, inflammatory bowel disease, immune disorders, inflammation, and cellular proliferation induced after medical procedures) that comprises a therapeutically effective amount of at least one compound of Formula I and at least one compound selected from: an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxic/cytostatic agent, an antiproliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, a PPAR-δ agonist, an inhibitor of cell proliferation and survival signaling, an agent that interfers with a cell cycle checkpoint, and an apoptosis inducing agent.
A preferred dosage is about 0.001 to 500 mg/kg of body weight/day of a compound of Formula I or a pharmaceutically acceptable salt or ester thereof. An especially preferred dosage is about 0.01 to 25 mg/kg of body weight/day of a compound of Formula I or a pharmaceutically acceptable salt or ester thereof.
The phrases “effective amount” and “therapeutically effective amount” mean that amount of a compound of Formula I, and other pharmacological or therapeutic agents described herein, that will elicit a biological or medical response of a tissue, a system, or a subject (e.g., animal or human) that is being sought by the administrator (such as a researcher, doctor or veterinarian) which includes alleviation of the symptoms of the condition or disease being treated and the prevention, slowing or halting of progression of one or more cellular proliferation diseases. The formulations or compositions, combinations and treatments of the present invention can be administered by any suitable means which produce contact of these compounds with the site of action in the body of, for example, a mammal or human.
For administration of pharmaceutically acceptable salts of the above compounds, the weights indicated above refer to the weight of the acid equivalent or the base equivalent of the therapeutic compound derived from the salt.
As described above, this invention includes combinations comprising an amount of at least one compound of Formula I or a pharmaceutically acceptable salt or ester thereof, and an amount of one or more additional therapeutic agents listed above (administered together or sequentially) wherein the amounts of the compounds/treatments result in desired therapeutic effect.
When administering a combination therapy to a patient in need of such administration, the therapeutic agents in the combination, or a pharmaceutical composition or compositions comprising the therapeutic agents, may be administered in any order such as, for example, sequentially, concurrently, together, simultaneously and the like. The amounts of the various actives in such combination therapy may be different amounts (different dosage amounts) or same amounts (same dosage amounts). Thus, for illustration purposes, a compound of Formula I and an additional therapeutic agent may be present in fixed amounts (dosage amounts) in a single dosage unit (e.g., a capsule, a tablet and the like). A commercial example of such single dosage unit containing fixed amounts of two different active compounds is VYTORIN® (available from Merck Schering-Plough Pharmaceuticals, Kenilworth, N.J.).
If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described herein and the other pharmaceutically active agent or treatment within its dosage range. Compounds of Formula I may also be administered sequentially with known therapeutic agents when a combination formulation is inappropriate. The invention is not limited in the sequence of administration; compounds of Formula I may be administered either prior to or after administration of the known therapeutic agent. Such techniques are within the skills of persons skilled in the art as well as attending physicians.
The pharmacological properties of the compounds of this invention may be confirmed by a number of pharmacological assays. The antitumor activity of the compounds of the present invention (including growth suppression activity as well as the intereference in the ability of tumerigenic cells to grow in the absence of adhesion) may be assayed by methods known in the art, for example, by using the methods as described in the examples (see for example, the proliferation assay and soft agar assay in the examples)
While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical composition. The compositions of the present invention comprise at least one active ingredient, as defined above, together with one or more acceptable carriers, adjuvants or vehicles thereof and optionally other therapeutic agents. Each carrier, adjuvant or vehicle must be acceptable in the sense of being compatible with the other ingredients of the composition and not injurious to the mammal in need of treatment.
Accordingly, this invention also relates to pharmaceutical compositions comprising at least one compound of Formula I, or a pharmaceutically acceptable salt or ester thereof and at least one pharmaceutically acceptable carrier, adjuvant or vehicle.
For preparing pharmaceutical compositions from the compounds described by this invention, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, cachets and suppositories. The powders and tablets may be comprised of from about 5 to about 95 percent active ingredient. Suitable solid carriers are known in the art, e.g., magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration. Examples of pharmaceutically acceptable carriers and methods of manufacture for various compositions may be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co., Easton, Pa.
The term pharmaceutical composition is also intended to encompass both the bulk composition and individual dosage units comprised of more than one (e.g., two) pharmaceutically active agents such as, for example, a compound of the present invention and an additional agent selected from the lists of the additional agents described herein, along with any pharmaceutically inactive excipients. The bulk composition and each individual dosage unit can contain fixed amounts of the afore-said “more than one pharmaceutically active agents”. The bulk composition is material that has not yet been formed into individual dosage units. An illustrative dosage unit is an oral dosage unit such as tablets, pills and the like. Similarly, the herein-described method of treating a subject by administering a pharmaceutical composition of the present invention is also intended to encompass the administration of the afore-said bulk composition and individual dosage units.
Additionally, the compositions of the present invention may be formulated in sustained release form to provide the rate controlled release of any one or more of the components or active ingredients to optimize the therapeutic effects. Suitable dosage forms for sustained release include layered tablets containing layers of varying disintegration rates or controlled release polymeric matrices impregnated with the active components and shaped in tablet form or capsules containing such impregnated or encapsulated porous polymeric matrices.
Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injection or addition of sweeteners and opacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.
Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas, e.g. nitrogen.
Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.
The compounds of the invention may also be deliverable transdermally. The transdermal compositions can take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.
The compounds of this invention may also be delivered subcutaneously.
Preferably the compound is administered orally.
Preferably, the pharmaceutical preparation is in a unit dosage form. In such form, the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active component, e.g., an effective amount to achieve the desired purpose.
The quantity of active compound in a unit dose of preparation may be varied or adjusted from about 1 mg to about 100 mg, preferably from about 1 mg to about 50 mg, more preferably from about 1 mg to about 25 mg, according to the particular application.
The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage regimen for a particular situation is within the skill of the art. For convenience, the total daily dosage may be divided and administered in portions during the day as required.
The amount and frequency of administration of the compounds of the invention and/or the pharmaceutically acceptable salts or esters thereof will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as severity of the symptoms being treated. A typical recommended daily dosage regimen for oral administration can range from about 1 mg/day to about 500 mg/day, preferably 1 mg/day to 200 mg/day, in two to four divided doses.
Another aspect of this invention is a kit comprising a therapeutically effective amount of at least one compound of Formula I or a pharmaceutically acceptable salt or ester thereof and at least one pharmaceutically acceptable carrier, adjuvant or vehicle.
Yet another aspect of this invention is a kit comprising an amount of at least one compound of Formula I or a pharmaceutically acceptable salt or ester thereof and an amount of at least one additional therapeutic agent listed above, wherein the amounts of the two or more ingredients result in desired therapeutic effect.
The invention disclosed herein is exemplified by the following preparations and examples which should not be construed to limit the scope of the disclosure. Alternative mechanistic pathways and analogous structures will be apparent to those skilled in the art.
The following solvents and reagents may be referred to by their abbreviations in parenthesis:
Thin layer chromatography: TLC
dichloromethane: CH2Cl2
ethyl acetate: AcOEt or EtOAc
methanol: MeOH
trifluoroacetate: TFA
triethylamine: Et3N or TEA
butoxycarbonyl: n-Boc or Boc
nuclear magnetic resonance spectroscopy: NMR
liquid chromatography mass spectrometry: LCMS
high resolution mass spectrometry: HRMS
milliliters: mL
millimoles: mmol
microliters: μl
grams: g
milligrams: mg
room temperature or rt (ambient): about 25° C.
dimethoxyethane: DME
Illustrating the invention are the following examples which, however, are not to be considered as limiting the invention to their details. Unless otherwise indicated, all parts and percentages in the following examples, as well as throughout the specification, are by weight.
2,4-Dichlorobenzoyl chloride (11.59 g, 7.76 mL, 55.3 mmoles) and N,O-dimethylhyoxy]amine hydrochloride (4.91 g, 50.3 mmoles) were dissolved in anhydrous dichloromethane (550 mL) and the mixture was cooled to 0° C. under argon. Anhydrous pyridine (8.76 g, 8.96 mL, 110.6 mmoles) was added dropwise to the stirred solution at 0° C. and the mixture was stirred at 0° C. for 6 h. The mixture was evaporated to dryness and the residue was partitioned between diethyl ether-dichloromethane (1:1) and brine. The organic layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (60×5 cm) using 1% (10% conc. NH4OH in methanol)-dichloromethane as the eluant to give the title compound as a colorless oil (11.78 g, 100%): ESMS: m/z234.0 (MH+); Found: C, 46.16; H, 3.55; Cl, 29.81; N, 5.96. C9H9Cl2NO2 requires: C, 46.18; H, 3.88; Cl, 30.29; N, 5.98; δH (CDCl3) 3.34 (3H, s, NCH3), 3.48 (3H, s, OCH3), 7.26 (2H, s, H3, H5) and 7.43 ppm (1H, s, H6); δC CDCl3) CH3: 32.3, 61.5; CH: 127.0, 128.7, 129.6; CH: 127.0, 128.7 129.6: C: 131.8, 133.8, 135.6, 165.1.
2,5-Dibromopyridine (10.2 g, 43.1 mmoles) was dissolved in anhydrous toluene (510 mL) and the mixture was stirred under argon at −78° C. 2.5M n-Butyl lithium in hexanes (20.3 mL, 51.72 mmoles) was added dropwise at −78° C. over 30 min and the mixture was stirred for 2 h at −78° C. A solution of 2,4-dichloro-N-methoxy-N-methylbenzamide (10.04 g, 43.1 mmoles) in anhydrous toluene (2 ml) was added dropwise to the stirred solution and the mixture was stirred at −78° C. for 1 h. The mixture was allowed to warm up to −10° C. Saturated aqueous NH4Cl (102 mL) was added and the mixture was stirred and allowed to warm up to 25° C. The toluene layer was separated and dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (30×5 cm) using 2% ethyl acetate in hexane as the eluant to give the title compound as a cream solid (9.42 g, 68%): FABMS: m/z 330 (MH+); HRFABMS: m/z 331.9066 (MH+). Calcd. for C12H7BrCl2NO: m/z 331.9065; Found: C, 43.27; H, 1.70; Br, 23.78, Cl, 21.79, N, 4.09; C12H6BrCl2NO requires: C, 43.54; H, 1.83; Br, 24.14, Cl, 21.42, N, 4.23; δH (CDCl3) 7.38 (1H, dd, H3′), 7.45 (2H, d, H5′ and H6′), 8.05 (2H, dd, H3 and H4) and 8.70 ppm (1H, s, H6); δC (CDCl3) CH: 124.9, 127.2, 130.0, 131.0, 140.0, 150.6; C, 125.6, 133.1, 136.2, 137.4, 151.8, 193.5, as well as the title compound of Preparative Example 3A (380.9 mg, 3%): FABMS: m/z 330.1 (MH+); Found: C, 43.80; H, 1.89; Br, 23.91; Cl, 21.82; N, 4.23. C12H6BrCl2NO requires: C, 43.54; H, 1.83; Br, 24.14; Cl, 21.42; N, 4.23; 8H (CDCl3) 7.38 (1H, s, H3′), 7.42 (1H, d, H5′), 7.53 (1H, d, H6′), 7.64 (1H, d, H5), 7.97 (1H, dd, H4) and 8.64 ppm (1H, d, H2); δC (CDCl3) CH: 127.8, 128.6, 130.5, 130.6, 138.8, 151.7/151.8; C, 131.1, 132.6, 135.4, 138.0, 147.7, 192.1 and bis-(2,4-dichlorophenyl)methanone (412.6 mg, 3%): FABMS: m/z 424 (MH+).
The title compound from Step A above (8.3 g, 25.1 mmoles) was dissolved in methanol (200 mL) and dichloromethane (50 mL) and cooled to 0° C. Sodium borohydride (1.38 g, 36.6 mmoles) was added and the mixture was stirred at 0° C. for 2.5 h and then allowed to warm up to 25° C. over a period of 1 h. The mixture was evaporated to dryness and the residue was partitioned between ethyl acetate and water. The ethyl acetate layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (30×5 cm) using 3-5% ethyl acetate in hexane as the eluant to give the title compound (6.93 g, 83%): FABMS: m/z 331.9 (MH+); δH (CDCl3) 6.18 (1H, d, CHOH), 7.17 (1H, d, H6′), 7.25 (1H, dd, H5′), 7.36 (1H, d, H3), 7.41 (1H, d, H3′), 7.77 (1H, dd, H4) and 8.63 ppm (1H, d, H6); δC (CDCl3) CH: 70.5, 122.6, 127.8, 129.4, 129.7, 139.8, 149.4; C, 119.9, 133.3, 134.4, 138.8, 158.2.
The title compound from Step D above (2.83 g, 8.57 mmoles) and triethylamine (3.58 mL, 25.7 mmoles) were added to anhydrous cyclohexane (50 mL) and the mixture was stirred at 25° C. for 15 min until all of the material had dissolved. Thionyl chloride (4.38 mL, 60 mmoles) was added and the mixture was stirred at 25° C. for 2.5 h. and then evaporated to dryness. the residue was chromatographed on a silica gel column (30×5 cm) using 2% ethyl acetate in hexane as the eluant to give 5-bromo-2-[chloro-(2,4-dichlorophenyl)methyl]pyridine (2.94 g, 98%).
2,5-Dibromopyridine (10.8 g, 45.6 mmoles) was dissolved in anhydrous diethyl ether (541 mL) and the mixture was stirred under argon at −78° C. 2.5M n-Butyl lithium in hexanes (21.5 mL, 54.7 mmoles) was added dropwise at −78° C. over 10 min and the mixture was stirred for 40 min at −78° C. A solution of 2,4-dichloro-N-methoxy-N-methylbenzamide (10.64 g, 45.61 mmoles) (prepared as described in Preparative Example 2, Step A above) in anhydrous diethyl ether (8 ml) was added dropwise over 10 min to the stirred solution and the mixture was stirred at −78° C. for 1 h. The mixture was allowed to warm up to −10° C. Saturated aqueous NH4Cl (108 mL) was added and the mixture was stirred and allowed to warm up to 25° C. The ether layer was separated and dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (30×5 cm) using 2% ethyl acetate in hexane as the eluant to give the title compound as a cream solid (10.11 g, 67%): FABMS: m/z 330.1 (MH+); Found: C, 43.80; H, 1.89; Br, 23.91; Cl, 21.82; N, 4.23. C12H6BrCl2NO requires: C43.54; H, 1.83; Br 24.14; Cl, 21.42; N, 4.23; δH (CDCl3) 7.38 (1H, d, H6′), 7.42 (1H, dd, H5′), 7.53 (1H, d, H3′), 7.64 (1H, d, H5), 7.97 (1H, dd, H4) and 8.64 ppm (1H, d, H2); δC (CDCl3) CH: 127.8, 128.6, 130.5, 130.6, 138.8, 151.7/151.8; C, 131.1, 132.6, 135.4, 138.0, 147.7, 192.1.
The title compound from Step A above (7.1 g, 21.5 mmoles) was dissolved in methanol (200 mL) and dichloromethane (50 mL) and cooled to 0° C. Sodium borohydride (1.18 g, 31.4 mmoles) was added and the mixture was stirred at 0° C. for 2.5 h and then allowed to warm up to 25° C. over a period of 1 h. The mixture was evaporated to dryness and the residue was partitioned between ethyl acetate and water. The ethyl acetate layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (30×5 cm) using 10% ethyl acetate in hexane as the eluant to give the title compound (6.88 g, 96%): FABMS: m/z 331.9 (MH+); Found: C, 43.32; H, 2.61; 23.33; Cl, 20.71: N, 3.96. C12H8BrCl2NO requires: C, 43.28; H, 2.42; Br, 23.99; Cl, 21.29; N, 4.21; δH (CDCl3) 6.17 (1H, d, CHOH), 7.32 (1H, d, H5′), 7.38 (1H, d, H3′), 7.46 (1H, d, H6′), 7.52 (1H, dd, H4), 7.57 (1H, d, H3) and 8.36 ppm (1H, d, H6); δC (CDCl3) CH: 69.6, 127.9, 128.1, 128.7, 129.6, 137.2, 149.0; C, 132.8, 134.7, 137.0, 138.5, 141.4.
The title compound from Step B above (3 g, 8.5 mmoles) and triethylamine (2.76 g, 3.8 mL, 25.5 mmoles) were dissolved in anhydrous cyclohexane (70 mL). Thionyl chloride (7.56 g, 4.64 mL, 59.5 mmoles) was added and the mixture was heated under nitrogen at 81° C. for 4 h. The mixture was evaporated to dryness and the residue was taken up in dichloromethane and chromatographed on a silica gel column (30×5 cm) using 3% ethyl acetate in hexane as the eluant to give 6-bromo-3-[chloro-(2,4-dichlorophenyl)methyl]pyridine as a red oil (2.97 g, 96%): FABMS: m/z 350.0 (MH+); HRFABMS: m/z 349.8908 (MH+), Calcd. for C12H8BrCl3N: m/z 349.8906; δH (CDCl3) 6.46 (1H, s, CHCl), 7.34 (1H, dd, H5′), 7.42 (1H, d, H3′), 7.48 (1H, d, H6′), 7.54 (1H, dd, H4), 7.58 (1H, d, H3) and 8.38 ppm (1H, d, H6); δC (CDCl3) CH: 56.5, 128.1, 128.2, 129.8, 130.5, 137.8, 149.4; C, 133.3, 134.9, 135.5, 135.5, 142.1.
3,5-Dichlorobenzoyl chloride (10.0 g, 47.7 mmoles) and N,O-dimethylhyoxylamine hydrochloride (4.23 g, 43.4 mmoles) were dissolved in anhydrous dichloromethane (475 mL) and the mixture was cooled to 0° C. under argon. Anhydrous pyridine (7.55 g, 7.79 mL, 95.5 mmoles) was added dropwise to the stirred solution at 0° C. and the mixture was stirred at 0° C. for 5 h. The mixture was evaporated to dryness and the residue was partitioned between diethyl ether-dichloromethane (1:1) and brine. The organic layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (60×5 cm) using 0.75% (10% conc. NH4OH in methanol)-dichloromethane as the eluant to give 3,5-dichloro-N-methoxy-N-methylbenzamide as a colorless oil (9.66 g, 95%): FABMS: m/z 234.2 (MH+); HRFABMS: m/z 234.0090 (MH+), Calcd. for C9H10Cl2NO2: m/z 234.0089; 8H (CDCl3) 3.34 (3H, s, NCH3), 3.54 (3H, s, OCH3), 7.44 (1H, dd, H4), 7.56 ppm (2H, d, H2 and H6); δC (CDCl3) CH3: 33.5, 61.5; CH: 126.9, 126.9, 130.6; C, 134.8, 134.8, 136.7, 166.8.
2,5-Dibromopyridine (9.21 g, 38.9 mmoles) was dissolved in anhydrous toluene (462 mL) and the mixture was stirred under argon at −78° C. 2.5M n-Butyl lithium in hexanes (18.66 mL, 46.7 mmoles) was added dropwise at −78° C. over 30 min and the mixture was stirred for 2 h at −78° C. A solution of 3,5-dichloro-N-methoxy-N-methylbenzamide (9.1 g, 38.9 mmoles) from Step A above, in anhydrous toluene (10 ml) was added dropwise to the stirred solution and the mixture was stirred at −78° C. for 1 h. The mixture was allowed to warm up to −10° C. Saturated aqueous NH4Cl (92 mL) was added and the mixture was stirred and allowed to warm up to 25° C. The toluene layer was separated and dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (45×8 cm) using 1% ethyl acetate in hexane as the eluant to give (5-bromopyridin-2-yl)-(3,5-dichlorophenyl)methanone as a cream solid (8.88 g, 69%): Found: C, 43.55; H, 1.88; Br, 24.13; Cl, 21.82; N, 4.22. C12H6BrCl2NO requires: C, 43.54; H, 1.83; Br, 24.14; Cl, 21.42; N, 4.23; FABMS: m/z 331.8 (MH+); HRFABMS: m/z 331.9066 (MH+), Calcd. for C12H7BrCl2NO: m/z 331.9065; δH (CDCl3) 7.57 (1H, dd, H4′), 7.97 (2H, d, H2 and H6′), 7.99 (1H, d, H3), 8.07 (1H, dd, H4) and 8.78 ppm (1H, d, H6); δC (CDCl3) CH: 126.1, 129.4, 129.4, 132.7, 140.2, 150.0; C, 125.4, 135.1, 135.1, 138.5, 152.1, 189.9.
The title compound from Step B above (8.18 g, 24.6 mmoles) was dissolved in methanol (200 mL) and dichloromethane (50 mL) and cooled to 0° C. Sodium borohydride (1.35 g, 35.9 mmoles) was added and the mixture was stirred at 0° C. for 2.5 h and then allowed to warm up to 25° C. over a period of 1 h. The mixture was evaporated to dryness and the residue was partitioned between ethyl acetate and water. The ethyl acetate layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (30×5 cm) using 4% ethyl acetate in hexane as the eluant to give (5-bromopyridin-2-yl)-(3,5-dichlorophenyl)methanol (8.06 g, 99%): Found: C, 43.20; H, 2.37; Br, 23.86; Cl, 21.69; N, 4.04. C12H8BrCl2NO requires; C, 43.28; H, 2.42; Br, 23.99; Cl, 21.29; N, 4.21; FABMS: m/z 334.0 (MH+); HRFABMS: m/z 333.9223 (MH+). Calcd. for C12H9BrCl2NO: m/z 333.9221; δH (CDCl3) 4.82 (1H, d, CHOH), 5.66 (1H, d, CHOH), 7.12 (1H, d, H3), 7.27 (2H, d, H2′ and H6′), 7.81 (1H, dd, H4) and 8.64 ppm (1H, d, H6); δC (CDCl3) CH: 73.9, 122.5, 125.4, 125.4, 128.3, 139.9, 149.5; C, 120.0, 135.3, 135.3, 146.0, 158.3.
The title compound from Step C above (0.303 g, 0.91 mmoles) and triethylamine (0.276 g, 0.38 mL, 2.73 mmoles) were dissolved in anhydrous cyclohexane (12 mL). Thionyl chloride (0.764 g, 0.465 mL, 6.37 mmoles) was added and the mixture was stirred under nitrogen at 25° C. for 3.5 h. The mixture was evaporated to dryness and the residue was taken up in dichloromethane and chromatographed on a silica gel column (30×2.5 cm) using 2% ethyl acetate in hexane as the eluant to give 5-bromo-2-[chloro-(3,5-dichlorophenyl)methyl]pyridine as an oil (0.314 g, 98%): FABMS: m/z 349.9 (MH+); HRFABMS: m/z 351.8881 (MH+). Calcd. for C12H8Cl3N: m/z 351.8881; δH (CDCl3) 5.99 (1H, s, CHCl), 7.30 (1H, dd, H4′), 7.34 (2H, d H2′ and H6′), 7.46 (1H, d, H3), 7.88 (1H, dd, H4) and 8.63 ppm (1H, d, H6); δC (CDCl3) CH: 62.2, 123.4, 126.4, 126.4, 128.7, 140.1, 150.6; C, 120.5, 135.3, 135.3, 142.7, 157.0.
Phenyl-(4-trifluoromethoxyphenyl)methanol (463.5 mg, 17.3 mmoles) [prepared by essentially the same procedure as described in Preparative Example 4, Step C by reduction of phenyl-(4-trifluoromethoxyphenyl)methanone. The latter may be prepared as described in: J. R. Desmurs, M. Labrouillere, C. Le Roux, H. Gaspard, A. Laporterie and J. Dubac, Tetrahedron Letters, 38(15), 8871-8874 (1997)] was dissolved in anhydrous toluene (10 mL) at 0° C. Thionyl chloride (0.882 mL, 12.1 mmoles) was added and the mixture was allowed to warm up to 25° C. over a period of 18 h. The solution was evaporated to dryness and the resulting material was azeotroped with anhydrous toluene to afford 4-trifluoromethoxybenzhydryl chloride, that was used without further purification in Example 10.
Ethyl 2-aminobenzoate (50 g, 44.76 mL, 302.7 mmoles) and chloroacetonitrile (68.56 g, 57.5 mL, 908.1 mmoles) were dissolved in anhydrous 1,4-dioxane (1L) and dry HCl gas was passed through the stirred solution at 25° C. for 5 h. The reaction was mildly exothermic for 4 h and after about 30 min the initial dense white precipitate dissolved. After about 1 h the mixture became turbid and a precipitate again formed. The reaction mixture was poured into ice/water (2L) and neutralized with concentrated ammonium hydroxide until pH 7.0 was reached. The resulting mixture was evaporated to dryness and the solid was triturated with distilled water, filtered off and rinsed with distilled water and then dried in vacuo at 50° C. for 18 h. The material was dissolved in 1,4-dioxane and silica gel was added. The mixture was then evaporated to dryness and the resulting solid was introduced onto a silica gel column (65×8.5 cm) and eluted with 3%-5%-10% methanol in dichloromethane to give 2-(chloromethyl)-3H-quinazolin-4-one (47.6 g, 81%): Found: C, 55.45; H, 3.47; N, 14.26. C9H7ClN2O requires: C, 55.54; H, 3.63; N, 14.39; FABMS: m/z 195.3 (MH+); δH (d6-DMSO) 4.53 (2H, s, CH2Cl), 7.51 (1H, ddd, H6), 7.64 (1H dd, H8), 8.00 (1H, ddd, H7) and 8.09 ppm (1H, dd, H5); δC (d6-DMSO)CH2: 43.2; CH: 125.9, 127.2, 127.2, 134.6; C, 121.2, 148.2, 152.3, 161.5.
4-Chloro-2-chloromethylquinazoline (20 g, 93.9 mmoles) [prepared as described by: C. J. Shishoo, M. B. Devani, V. S. Bhadti, K. S. Jain and S. Anathan, J. Heterocyclic Chem., 27, 119-126 (1990)], L-(+)-valine methyl ester hydrochloride (15.74 g, 93.9 mmoles) and potassium carbonate (14.28 g, 103.3 mmoles) were added to anhydrous acetonitrile (700 mL) and the mixture was heated under reflux and under nitrogen at 80° C. for 18 h. The mixture was evaporated to dryness and the residue was partitioned between dichloromethane and saturated aqueous sodium bicarbonate. The organic layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (60×8.5 cm) using 2% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-(+)-(2-chloromethylquinazolin-4-ylamino)-3-methylbutyric acid methyl ester (23.54 g, 81%): FABMS: m/z 308.2 (MH+); HRFABMS: m/z 308.1161 (MH+). Calcd. for C15H19ClN3O2: m/z 308.1166; δH (CDCl3) 1.03 (3H, d, CH(CH3)2), 1.08 (3H, d, CH(CH3)2), 2.37 (3H, dq, CH(CH3)2), 3.81 (3H, s, COOCH3), 4.61 (2H, s, CH2Cl), 5.08 (1H, dd, CHCH(CH3)2), 6.42 (1H, d, NH), 7.44 (1H, ddd, H6), 7.73 (1H, ddd, H7), 7.77 (1H, dd, H5) and 7.80 ppm (1H, dd, H8); δC (CDCl3) CH3: 18.6, 19.1, 58.6; CH2: 48.2; CH: 31.5, 52.4, 120.6, 126.5, 128.5, 133.0; C, 113.5, 149.9, 159.8, 161.3, 173.4; [α]D25° C.−22.7° (c=0.51, MeOH).
2(S)-(+)-(2-Chloromethylquinazolin-4-ylamino)-3-methylbutyric acid methyl ester (1.79 g, 5.82 mmoles) (prepared as described in Preparative Example 9), dibenzylamine (1.15 g, 1.12 mL, 6.69 mmoles) and anhydrous potassium carbonate (884 mg, 6.40 mmoles) were added to anhydrous acetonitrile (150 mL) and the mixture was heated under reflux and under nitrogen at 80° C. for 18 h. The mixture was evaporated to dryness and the residue was partitioned between dichloromethane and saturated aqueous sodium bicarbonate. The organic layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (30×5 cm) using 1% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-(−)-{2-[(dibenzylaminomethyl)quinazolin-4-yl]amino}-3-methylbutyric acid methyl ester (2.7 g, 99%): FABMS: m/z 469.3 (MH+); HRFABMS: m/z 469.2598 (MH+). Calcd. for C29H33N4O2: m/z 469.2604; δH (CDCl3) 1.08 (3H, d, CH(CH3)2), 1.14 (3H, d, CH(CH3)2), 2.43 (1H, dq, CH(CH3)2), 3.80 (3H, s, COOCH3), 3.83 (6H, s, —CH2N(CH2C6H5)2), 5.28 (1H, m, CHCH(CH3)2), 6.21 (1H, d, NH), 7.23, 7.30, 7.49, 7.73, 7.80 and 7.84 ppm (14H, m, H6, H7, H4, H8 and CH2C6H5); δC (CDCl3) CH3: 18.6, 19.0, 58.3; CH2: 57.8, 57.8, 60.0; CH: 31.5, 52.3, 120.6, 125.7, 126.7, 126.7, 128.1, 128.4, 128.4, 128.4, 128.4, 129.0, 129.0, 129.0, 129.0; C, 113.5, 139.9, 139.9, 149.7, 159.2, 164.3 173.2; [α]D25° C.−18.4° (c=0.50, MeOH).
2(S)-(−)-{2-[(Dibenzylaminomethyl)quinazolin-4-yl]amino}-3-methylbutyric acid methyl ester (20 g, 42.69 mmoles) (prepared as described in Preparative Example 10), ammonium formate (13.46 g, 213.4 mmoles) and 10% Pd—C (50% wet with water) (40 g wet equivalent to 20 g dry weight) were added to methanol (1L) under nitrogen and the mixture was heated under reflux at 87° C. for 3 h. The catalyst was filtered off through Celite© and the latter was washed with methanol. The combined filtrates were evaporated to dryness and the residue was chromatographed on a silica gel column (60×5 cm) using 3% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-(−)-(2-aminomethylquinazolin-4-ylamino)-3-methylbutyric acid methyl ester (7.16 g, 58%): FABMS: m/z 289.0 (MH+), HRFABMS: m/z 289.1663 (MH+). Calcd for C15H21N4O2: m/z 289.1665; δH (CDCl3) 1.02 (3H, d, CH(CH3)2), 1.08 (3H, d, CH(CH3)2), 2.35 (1H, dq, CH(CH3)2), 2.52 (2H, bs, NH2), 3.77 (3H, s, COOCH3), 3.96 (2H, s, CH2NH2), 5.03 (1H, m, CHCH(CH3)2), 6.26 (1H, bs, NH), 7.43 (1H, ddd, H6), 7.72 (1H, ddd, H7) and 7.77 ppm (2H, dd, H5 and H8); δC (CDCl3) CH3: 18.6, 19.1, 58.6; CH2: 48.5; CH: 31.5, 52.3, 120.7, 125.6, 128.2, 132.9; C, 113.5, 149.9, 159.4, 165.9, 173.3; [α]D25° C.−39.8° (c=0.51, MeOH).
4-Chloro-2-chloromethylquinazoline (30 g, 97.5 mmoles) [prepared as described by: C. J. Shishoo, M. B. Devani, V. S. Bhadti, K. S. Jain and S. Anathan, J. Heterocyclic Chem., 27, 119-126 (1990), L-(+)-valinamide hydrochloride (21.5 g, 140.9 mmoles) and potassium carbonate (42.8 g, 309.7 mmoles) were added to anhydrous acetonitrile (500 mL) and the mixture was heated under reflux and under argon at 80° C. for 24 h. The mixture was evaporated to dryness and the residue was partitioned between dichloromethane and water. The organic layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (60×8.5 cm) using 2% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-(−)-(2-aminomethylquinazolin-4-ylamino)-3-methylbutyramide (22.34 g, 78%): FABMS: m/z 293.0 (MH+); HRFABMS: m/z 293.1166 (MH+). Calcd. for C14H18ClN4O: m/z 293.1169; 8H (CD3OD) 1.09 (6H, d, CH(CH3)2), 2.12 (3H, dq, CH(CH3)2), 4.57 (2H, s, CH2Cl), 4.77 (1H, d, CHCH(CH3)2), 4.87 (3H, s, NH and NH2), 7.49 (1H, ddd, H6), 7.68 (1H, ddd, H7), 7.75 (1H, dd, H5) and 8.18 ppm (1H, dd, H8); δC (CD3OD) CH3: 19.6, 19.6; CH2: obscured under MeOH; CH: 31.5, 61.7, 123.4, 127.7, 127.7, 134.4; C, 114.8, 150.4, 162.1, 163.1, 177.0; [α]D25° C.−10.6° (c=1.01, MeOH).
4-Chloro-2-chloromethylquinazoline (6.39 g, 20.76 mmoles) [prepared as described by: C. J. Shishoo, M. B. Devani, V. S. Bhadti, K. S. Jain and S. Anathan, J. Heterocyclic Chem., 27, 119-126 (1990), L-(+)-isoleucinamide hydrochloride (5 g, 20.76 mmoles) and potassium carbonate (4.56 g, 20.76 mmoles) were added to anhydrous acetonitrile (250 mL) and the mixture was heated under reflux and under argon at 80° C. for 25 h. Additional potassium carbonate (4.56 g, 20,76 mmoles) was added together with absolute ethanol (50 mL) and the slurry was stirred at 80° C. for a total of 46 h. The mixture was evaporated to dryness and the residue was partitioned between dichloromethane and saturated aqueous sodium bicarbonate. The organic layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (60×5 cm) using 2% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2-chloromethyl-4-ethoxyquinazoline (452.3 mg, 7%): Found: C, 59.27; H, 5.01; Cl, 15.93; N, 12.58. C11H11ClN2O requires: C, 59.33; H, 4.98; Cl, 15.92; N, 12.58; FABMS: m/z 222.9 (MH+); δH (CDCl3) 1.52 (3H, dd, OCH2CH3), 4.63 (2H, q, OCH2CH3′), 4.72 (2H, d, CH2Cl), 7.55 (1H, ddd, H6′), 7.81 (1H, ddd, H7) 7.90 (1H, dd, H5) and 8.14 ppm (1H dd, H8); δC (CDCl3) CH3: 14.4; CH2: 47.8, 63.5; CH: 123.6, 127.3, 127.6, 133.8; C, 115.4, 151.0, 161.2, 167.5 and 2(S)-(−)-(2-chloromethylquinazolin-4-ylamino)-3(R)-methylpentanamide (5.33 g, 58%): FABMS: m/z 307.0 (MH+); HRFABMS: m/z 307.1323 (MH+). Calcd. for C15H20ClN4O: m/z 307.1326; δH (CDCl3) 0.93 (3H t, CH3CHCH2CH3), 1.05 (3H, d, CH3CHCH2CH3), 1.30 (1H, dq, CH3CHCH2CH3), 1.70 (1H, dq, CH3CHCH2CH3), 2.24 (1H, ddq, CH3CHCH2CH3), 4.62 (2H, s, —CH2Cl), 4.73 (1H, dd, NHCHCONH2), 6.11 (1H, bs, NH), 6.57 (1H, bs NH2), 7.24 (1 h, bs, NH2), 7.34 (1H, ddd, H6), 7.64 (1H, ddd, H7), 7.70 (1H, dd, H5) and 7.77 ppm (1H, dd, H8); δC (CDCl3) CH3: 11.0, 15.7; CH2: 25.5, 47.9; CH: 35.9, 59.1, 121.2, 126.7, 127.6, 133.3; C, 113.4, 148.5, 160.0, 160.7, 174.6; [α]D25° C.−18.9° (c=0.53, MeOH).
4-Chloro-2-chloromethylquinazoline (2 g, 6.5 mmoles) [prepared as described by: C. J. Shishoo, M. B. Devani, V. S. Bhadti, K. S. Jain and S. Anathan, J. Heterocyclic Chem., 27, 119-126 (1990), L-(+)-leucinamide hydrochloride (1.56 g, 6.5 mmoles) and potassium carbonate (1.43 g, 6.5 mmoles) were added to anhydrous acetonitrile (50 mL) and the mixture was heated under reflux and under argon at 80° C. for 18 h. The mixture was evaporated to dryness and the residue was partitioned between dichloromethane and water. The organic layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (30×5 cm) using 2% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-(+)-(2-chloromethylquinazolin-4-ylamino)-4-methylpentanamide (1.69 g, 59%): FABMS: m/z 307.1 (MH+), HRFABMS: m/z 307.1321 (MH+).
Calcd. for C15H20ClN4O: m/z 307.1326; δH (CDCl3) 0.97 (3H, s, CH(CH3)2), 1.02 (3H, s, CH(CH3)2), 1.83 (1H, m, CH2CH(CH3)2), 1.92 (2H, m, CH2CH(CH3)2), 4.63 (2H, m, CH2Cl), 4.97 (1H, m, NHCHCONH2), 5.84 (1H, bs, NH), 6.74 (1H, bs, CONH2), 6.91 (1H, bs, CONH2), 7.32 (1H, m, H6), 7.66 ppm (3H, m, H5′ H7 and H8); δC (CDCl3) CH3: 22.3, 23.1; CH2: 44.4, 48.4; CH: 25.0, 52.7, 120.9, 126.6, 128.1, 133.1; C, 113.4, 149.4, 159.9, 160.7, 175.4; [α]D25° C.+5.4° (c=0.53, MeOH).
2(S)-(−)-(2-Chloromethylquinazolin-4-ylamino)-3-methylbutyramide (16.2 g, 55.3 mmoles) (prepared as described in Preparative Example 12), dibenzylamine (15.12 g, 14.74 mL, 76.4 mmoles) and anhydrous potassium carbonate (11.63 g, 84.1 mmoles) were added to anhydrous acetonitrile (700 mL) and the mixture was heated under reflux and under nitrogen at 80° C. for 18 h. The mixture was evaporated to dryness and the residue was partitioned between dichloromethane and saturated aqueous sodium bicarbonate. The organic layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (60×5 cm) using 3% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-(−)-{2-[(dibenzylamino)methyl]quinazolin-4-ylamino}-3-methylbutyramide (20.65 g, 82%): FABMS: m/z 454.34 (MH+); HRFABMS: m/z 454.2611. Calcd. for C28H32N5O: m/z 454.2607; δH (CDCl3) 1.08 (3H, d, CH(CH3)2), 1.10 (3H, d, CH(CH3)2), 2.43 (1H, dq, CH(CH3)2), 3.78 (6H, s, CH2N(CH2C6H5)2), 4.71 (1H, m, NH), 5.62 (1H, m, —CHCH(CH3)2), 6.55 (2H, m, NH2), 7.23, 7.30, 7.47, 7.68 and 7.78 ppm (14H, m, H6, H7, H4, H8 and CH2C6H5); δC (CDCl3) CH3: 19.0, 19.6; CH2: 58.3, 58.3, 60.4; CH: 29.8, 59.9, 120.9, 125.7, 127.0, 127.0, 128.3, 128.5, 128.5, 128.5, 128.5, 129.0, 129.0, 129.0, 129.0; C, 113.6, 139.7, 139.7, 149.9, 159.6, 164.0, 174.3; [α]D25° C.−8.6° (c=0.52, MeOH).
2(S)-(−)-{2-[(Dibenzylamino)methyl]quinazolin-4-ylamino}-3-methylbutyramide (18 g, 39.68 mmoles) (prepared as described in Preparative Example 15), ammonium formate (12.5 g, 198.4 mmoles) and 10% Pd—C (50% wet with water) (36 g wet equivalent to 18 g dry weight) were added to methanol (500 mL) and ethyl acetate (300 mL) under argon and the mixture was heated under reflux at 87° C. for 3 h. The catalyst was filtered off through Celite© and the latter was washed with methanol. The combined filtrates were evaporated to dryness and the residue was chromatographed on a silica gel column (60×5 cm) using 10% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-(+)-(2-aminomethylquinazolin-4-ylamino)-3-methylbutyramide (6.84 g, 62%): FABMS: m/z 274.1 (MH+), HRFABMS: m/z 274.1669 (MH+). Calcd for C14H20N5O: m/z 274.1668; δH (CDCl3) 1.01 (3H, d, CH(CH3)2), 1.02 (3H, d, CH(CH3)2), 2.22 (1H, dq, CH(CH3)2), 3.15 (4H, s, NH2), 3.37 (1H, s, NH), 3.87 (2H, s, CH2NH2), 4.78 (1H, d, CHCH(CH3)2), 7.36 (1H, ddd, H6), 7.39 (1H, ddd, H7), 7.66 (1H, dd, H5) and 7.89 ppm (1H, dd, H8); δC (CDCl3) CH3: 18.8, 19.3; CH2: 47.6; CH: 30.8, 59.4, 121.5, 125.8, 127.1, 133.1; C, 113.5, 149.4, 159.9, 164.7, 175.1; [α]D25° C.+2.3° (c=0.70, MeOH).
2-(Chloromethyl)-3H-quinazolin-4-one (2.2 g, 10.3 mmoles) (prepared as described in Preparative Example 6), N-methylbenzylamine (4.38 mL, 30.9 mmoles) and anhydrous potassium carbonate (1.72 g, 11.33 mmoles) were added to 200 proof ethanol (40 mL) and the mixture was stirred at 25° C. for 23 h. The mixture was evaporated to dryness and the residue was triturated with hexane and filtered. The insoluble solid was chromatographed on a silica gel column (30×9 cm) using 1.5% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2-[(benzylmethylamino)methyl]-3H-quinazolin-4-one (1.58 g, 50%): Found: C, 72.88; H, 6.24; N, 14.94. C17H17N3O requires: C, 73.10; H, 6.13; N, 15.04; FABMS: m/z 280.0 (MH+), HRFABMS: m/z 280.1438 (MH+). Calcd. for C17H18N3O: 280.1450; δH (CDCl3) 2.33 (3H, s, NCH3), 3.57 (2H, s, CH2N), 3.67 (2H, s, CH2N), 7.27 (1H, m, C6H5CH2), 7.34 (4H, m, C6H5CH2), 7.44 (1H, ddd, H6), 7.62 (1H, dd, H8), 7.73 (1H, ddd, H7) and 8.26 ppm (1H, dd, H5); δC (CDCl3) CH3: 43.1; CH2: 59.1, 62.4; CH: 126.7, 126.7, 127.0, 127.9, 128.7, 128.7, 129.2, 129.2, 134.7; C, 121.8, 137.2, 148.9, 154.0, 161.6. The compound was found to have % Residual T @ 2 ug/mL according to scintillation proximity assay (SPA) of “B” (see description of assays below).
2-[(Benzylmethylamino)methyl]-3H-quinazolin-4-one (0.5 g, 1.79 mmoles) (prepared as described in Preparative Example 17) was dissolved in phosphorous oxychloride (1.67 mL, 17.9 mmoles) and the mixture was heated under reflux at 110° C. for 2 h and the allowed to cool to 25° C. for 1 h. The mixture was evaporated to dryness and the residue was taken up in dichloromethane and washed with saturated aqueous sodium bicarbonate. The organic layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (60×2.5 cm) using 20% ethyl acetate in hexane as the eluant to give N-benzyl-N-[(4-chloroquinazolin-2-yl)methyl]methylamine (230.8 mg, 43%): FABMS: m/z 297.9 (MH+); HRFABMS: m/z 298.1109 (MH+). Calcd. for C17H17ClN3: m/z 298.1111; δH (CDCl3) 2.43 (3H, s, NCH3), 3.74 (2H, s, CH2N), 4.00 (2H, s, CH2N), 7.20-7.32 (3H, m, C6H5CH2), 7.42 (2H, d, C6H5CH2), 7.68 (1H, ddd, H6), 7.93 (1H, ddd, H7), 8.10 (1H, dd, H5) and 8.23 ppm (1H, dd, H8); δC (CDCl3) CH3: 42.8; CH2: 61.9, 63.3; CH: 125.8, 127.2, 128.5, 128.7, 129.5, 129.5, 134.9; C, 122.5, 138.3, 151.4, 162.6, 163.6.
N-Benzyl-N-[(4-chloroquinazolin-2-yl)methyl]methylamine (14.55 g, 48.9 mmoles) (prepared as described in Preparative Example 18) and 3-dimethylaminopropylamine (12.3 mL, 97.7 mmoles) were dissolved in 200 proof ethanol (500 mL) and the mixture was heated under reflux at 80° C. for 18 h. The mixture was evaporated to dryness and the residue was chromatographed on a silica gel column (45×8 cm) using 6% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give N,N-dimethyl-N′-[2-{(N-methyl-N-benzylamino)methyl}quinazolin-4-yl]propane-1,3-diamine (17.46 g, 98%): FABMS: m/z 364.3 (MH+); HRFABMS: m/z 364.2497 (MH+). Calcd. for C22H30N5: m/z 364.2501; δH (CDCl3) 1.85 (2H, m, NHCH2CH2CH2N(CH3)2), 2.38 (6H, s, NHCH2CH2CH2N(CH3)2), 2.40 (3H, s, CH2N(CH3)CH2), 2.59 (2H, dd, NHCH2CH2CH2N(CH3)2), 3.77 (2H, s, CH2N(CH3)CH2), 3.77 (2H, m, NHCH2CH2CH2N(CH3)2), 3.79 (2H, s, CH2N(CH3)CH2), 7.18-7.32 (3H, m, C6H5CH2), 7.37 (1H, ddd, H6), 7.43 (2H, d, C6H5CH2), 7.59 (1H, dd, H5), 7.66 (1H, ddd, H7), 7.83 (1H, d, H8) and 8,60 ppm (1H, dd, NHCH2CH2CH2N(CH3)2)); δC (CDCl3) CH3: 42.7, 45.5, 45.5: CH2: 24.7, 42.4, 59.8, 61.7, 64.3; CH: 121.0, 125.2, 126.8, 128.1, 128.1, 128.3, 129.4, 129.4, 132.1; C, 114.2, 139.3, 149.9, 159.9, 164.4. The compound was found to have % Residual T @ 2 ug/mL according to scintillation proximity assay (SPA) of “B” (see description of assays below).
N,N-Dimethyl-N′-[2-{(N-methyl-N-benzylamino)methyl}quinazolin-4-yl]propane-1,3-diamine (8 g, 22.0 mmoles) (prepared as described in Preparative Example 19) and ammonium formate (6.94 g, 110 mmoles) were dissolved in methanol (520 mL). 10% Pd—C catalyst (8.4 g) was added under argon and the mixture was heated under reflux at 87° C. for 1.25 h. The catalyst was filtered off using Celite© and the latter was washed with methanol. The combined filtrates were evaporated to dryness and the residue was chromatographed on a silica gel column (50×8 cm) using 8% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give N,N′-dimethyl-N′-[2-(methylaminomethyl)quinazolin-4-yl]propane-1,3-diamine (5.02 g, 83%): FABMS: m/z 274.2 (MH+); HRFABMS: m/z 274.2030 (MH+). Calcd. for C15H24N5: m/z 274.2032; δH (CDCl3) 1.84 (2H, m, NHCH2CH2CH2N(CH3)2), 2.37 (6H, s, NHCH2CH2CH2N(CH3)2), 2.54 (3H, s, CH2NH(CH3)), 2.60 (2H, dd, NHCH2CH2CH2N(CH3)2), 3.59 (1H, bs, CH2NH(CH3)), 3.75 (2H, m, NHCH2CH2CH2N(CH3)2), 3.90 (2H, s, CH2NH(CH3)), 7.37 (1H, ddd, H6), 7.58 (1H, dd, H5), 7.64 (1H, ddd, H7), 7.74 (1H, dd, H8) and 8,70 ppm (1H, dd, NHCH2CH2CH2N(CH3)2)); δC (CDCl3) CH3: 36.1, 45.5, 45.5; CH2: 24.6, 42.4, 57.6, 59.8; CH: 121.1, 125.2, 127.9, 132.3; C, 114.3, 149.7, 159.9, 164.3. The compound was found to have % Residual T @ 2 ug/mL according to scintillation proximity assay (SPA) of “C” (see description of assays below).
N-(tert-Butoxycarbonyl)-L(−)-valine (19, 4.58 mmoles), methoxylamine hydrochloride (499.7 mg, 5.98 mmoles), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (1.15 g, 5.98 mmoles), hydroxybenzotriazole (808.5 mg, 5.98 mmoles) and N-methylmorpholine (1.21 g, 1.316 mL, 11.91 mmoles) were dissolved in anhydrous DMF (20 mL) and the mixture was stirred at 25° C. for 89 h. The solution was evaporated to dryness and the residue was taken up in dichloromethane and washed with saturated aqueous sodium bicarbonate. The organic layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (60×2.5 cm) using 0.3%-3% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give [1(S)-(−)-methoxycarbamoyl-2-methylpropyl]carbamic acid tert-butyl ester (857.7 mg, 76%): FABMS: m/z 247.4 (MH+); Found: C, 54.03; H, 9.18; N, 11.38; C11H22N2O4 requires: C, 53.64; H, 9.00; N, 11.37; δH (CDCl3) 0.96 (6H, d, CHCH(CH3)2), 1.43 (9H, s, NHCOOC(CH3)3), 2.05 (1H, dq, CHCH(CH3)2), 3.76 (1H, bs, NH), 3.76 (3H, s, CONHOCH3), 5.23 (1H, m, CHCH(CH3)2) and 9.61 ppm (1H, bs, NH); δC (CDCl3) CH3: 18.5, 19.2, 28.4, 28.4, 28.4, 57.8; CH: 30.8, 64.3; C, 80.4, 156.1, 165.2, 169.2; [α]D25° C.−32.7° (c=1.02, MeOH).
[1 (S)-(−)-Methoxycarbamoyl-2-methylpropyl]carbamic acid tert-butyl ester (812 mg, 3.3 mmoles) (prepared as described in Preparative Example 27, Step A above) was dissolved in methanol (10 mL) and 10% concentrated sulfuric acid in 1,4-dioxane (v/v) (10 mL) was added. The mixture was stirred at 25° C. for 4 h. The reaction was diluted with methanol and BioRad® AG1X8 (OH−) resin was added until the pH reached 10. The resin was filtered off and washed with methanol. The combined filtrates were evaporated to dryness the residue was chromatographed on a silica gel column (30×2.5 cm) using 5% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give (S)-(+)-amino-N-methoxy-3-methylbutyramide (172.7 mg, 48%): FABMS: m/z 147.2 (MH+); Found: C, 49.04; H, 9.39; N, 18.65; C6H14N2O2 requires; C, 49.30; H, 9.65; N, 19.16; δH (CDCl3) 0.87 (6H, d, CHCH(CH3)2), 0.98 (9H, s, NHCOOC(CH3)3), 1.40 (2H, bs, NH2), 2.29 (1H, dq, CHCH(CH3)2), 3.25 (1H, d, CHCH(CH3)2) and 3.78 (3H, s, CONHOCH3); δC (CDCl3) CH3: 16.2, 19.4, 64.5; CH: 31.0, 59.5; C, 171.5; [α]D25° C.+39.5° (c=0.53, MeOH).
N-(tert-Butoxycarbonyl)-L(−)-valine (1 g, 4.58 mmoles), ethoxy]amine hydrochloride (583.7 mg, 5.98 mmoles), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (1.15 g, 5.98 mmoles), hydroxybenzotriazole (808.5 mg, 5.98 mmoles) and N-methylmorpholine (1.21 g, 1.316 mL, 11.91 mmoles) were dissolved in anhydrous DMF (20 mL) and the mixture was stirred at 25° C. for 89 h. The solution was evaporated to dryness and the residue was taken up in dichloromethane and washed with saturated aqueous sodium bicarbonate. The organic layer was dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (60×2.5 cm) using 0.3%-3% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give [1 (S)-(−)-ethoxycarbamoyl-2-methylpropyl]carbamic acid tert-butyl ester (934.1 mg, 78%): FABMS: m/z 261.3 (MH+); Found: C, 55.83; H, 9.28; N, 10.78; C12H24N2O4 requires: C, 55.36; H, 9.29; N, 10.76; δH (CDCl3) 0.96 (6H, d, CHCH(CH3)2), 1.27 (3H, t, OCH2CH3), 1.43 (9H, s, NHCOOC(CH3)3), 2.06 (1H, dq, CHCH(CH3)2), 3.73 (1H, t, NH), 3.95 (2H, q, CONHOCH2CH3), 5.18 (1H, d, CHCH(CH3)2) and 9.32 ppm (1H, bs, NH); δC (CDCl3) CH3: 13.5, 18.5, 19.2, 28.4, 28.4, 28.4; CH2: 72.2; CH: 30.7, 57.9; C, 80.3, 156.1, 169.2; [α]D25° C.−35.9° (c=1.05, MeOH).
[1 (S)-(−)-Ethoxycarbamoyl-2-methylpropyl]carbamic acid tert-butyl ester (894 mg, 3.4 mmoles) (prepared as described in Preparative Example 28, Step A above) was dissolved in methanol (10 mL) and 10% concentrated sulfuric acid in 1,4-dioxane (v/v) (10 mL) was added. The mixture was stirred at 25° C. for 4 h. The reaction was diluted with methanol and BioRad® AG1X8 (OH−) resin was added until the pH reached 10. The resin was filtered off and washed with methanol. The combined filtrates were evaporated to dryness the residue was chromatographed on a silica gel column (30×2.5 cm) using 10% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give (S)-(+)-amino-N-ethoxy-3-methylbutyramide (352 mg, 64%): FABMS: m/z 161.3 (MH+); Found: C, 52.75; H, 9.84; N, 17.33; C7H16N2O2 requires; C, 52.48; H, 10.07; N, 17.49; δH (CDCl3) 0.87 (6H, d, CHCH(CH3)2), 0.97 (9H, s, NHCOOC(CH3)3), 1.27 (3H, t, CONHOCH2CH3), 1.36 (2H, bs, NH2), 2.26 (1H, dq, CHCH(CH3)2), 3.24 (1H, d, CHCH(CH3)2), 3.97 (2H, q, CONHOCH2CH3) and 9.57 ppm (1H, bs, NH); δC (CDCl3) CH3: 13.5, 16.3, 19.4, 64.5; CH2: 72.2; CH: 31.0, 59.5; C, 171.6; [α]D25° C.+33.9° (c=0.51, MeOH).
Using the procedure described in the literature for the conversion of racemic 2,4-diaminobutyric acid into racemic 4-benzyloxycarbonylamino-2-tert-butoxycarbonylaminobutyric acid [A. D. Borthwick, S. J. Angier, A. J. Crame, A. M. Exall, T. M. Haley, G. J. Hart, A. M. Mason, A. M. K. Pennell and G. G. Weingarten, J. Med. Chem., 43(23), 4452-4464 (2000)], 2(S)-(−)-2,4-diaminobutyric acid (20.78 g, 108 mmoles) was converted into 4-benzyloxycarbonylamino-2(S)-(−)-tert-butoxycarbonylaminobutyric acid (17.07 g, 69%): FABMS: m/z 353.0 (MH+); δH (CDCl3) 1.43 (9H, s, COOC(CH3)3), 5.04/5.13 (2H, AB system, CH2C6H5) and 7.37 ppm (5H, m, CH2C6H5); δH (CDCl3) CH3: 28.4, 28.4, 28.4; CH2: 33.4, 37.2, 67.1; CH: 50.9, 128.2, 128.2, 128.6, 128.6, 128.6; C, 80.5, 136.4, 156.0, 157.1, 176.0; [α]D25° C.−13.5° (c=0.51, MeOH). The (S)-(−)-Isomer has also been prepared by an alternative procedure [K. Vogler, R. O. Studer, P. Lanz, W. Lergier and E. Bohni, Helv. Chim. Acta, 48(5), 1161-1177 (1965)].
4-Benzyloxycarbonylamino-2(S)-(−)-tert-butoxycarbonylaminobutyric acid (17 g, 48.2 mmoles) and 37% aqueous formaldehyde (9.03 mL, 115.8 mmoles) were dissolved in methanol-distilled water (1:1) (260 mL). 10% Pd—C (wet; ˜7 g) was added under argon and the mixture was hydrogenated at 25° C. and 50 psi on a Parr hydrogenator for 74 h. The catalyst was filtered off through Celite© and the latter was washed with methanol-distilled water (1:1). The combined filtrates were evaporated to dryness to give 2(S)-(−)-tert-butoxycarbonylamino-4-dimethylaminobutyric acid (10.89 g, 92%): FABMS: m/z 247.0 (MH+); HRFABMS: m/z 247.1660 (MH+). Calcd. for C11H23N2O4: m/z 247.1658; δH (CDCl3) 1.34 (9H, s, COOC(CH3)3), 1.80 (1H, m, CHCH2CH2N(CH3)2), 1.87 (1H, m, CHCH2CH2N(CH3)2), 2.43 (6H, s, N(CH3)2), 2.68 (1H, m, CHCH2CH2N(CH3)2), 2.79 (1H, m, CHCH2CH2N(CH3)2), 3.74 (1H, m, CHCH2CH2N(CH3)2) and 6.47 ppm (1H, d, NH); δC (CDCl3) CH3: 28.3, 28.3, 28.3, 42.7, 42.7; CH2: 28.3, 56.2; CH: 53.9; C, 79.5, 155.8, 175.2; [α]D25° C.−1.7° (c=0.30, MeOH).
2(S)-(−)-tert-Butoxycarbonylamino-4-dimethylaminobutyric acid (5 g, 20.3 mmoles) (prepared as described in Preparative Example 29, step B above), N-methylmorpholine (2.26 g, 2.46 mL, 22.3 mmoles) and isobutyl chloroformate (3.05 g, 2.9 mL, 22.3 mmoles) were dissolved in anhydrous THF (200 mL) and the mixture was stirred at 0° C. for 1.5 h. Conc. ammonium hydroxide (30%) (10 mL) was added and the mixture was stirred at 0° C. for 3 h. The mixture was evaporated to dryness and the product was chromatographed on a silica gel column (30×5 cm) using 1% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-tert-butoxycarbonylamino-4-dimethylaminobutyric acid isobutyl ester (3.56 g, 58%): FABMS: m/z 303.1 (MH+); HRFABMS: m/z 303.2287 (MH+). Calcd. for C15H31N2O4: m/z 303.2284; δH (CDCl3) 0.93 (6H, d, COOCH2CH(CH3)2), 1.42 (9H, s, COOC(CH3)3), 1.83 (1H, m, OCH2CH(CH3)2), 1.92 (1H, m, CHCH2CH2N(CH3)2), 1.97 (1H, m, CHCH2CH2N(CH3)2), 2.22 (6H, s, N(CH3)2), 2.31 (1H, m, CHCH2CH2N(CH3)2), 2.40 (1H, m, CHCH2CH2N(CH3)2), 3.38 (2H, m, OCH2CH(CH3)2), 4.33 (1H, m, CHCH2CH2N(CH3)2) and 5.90 ppm (1H, m, NH); δC (CDCl3) 19.1, 19.1, 28.4, 28.4, 28.4, 45.4, 45.4; CH2: 29.5, 56.0, 71.3; CH: 27.8, 53.0; C, 79.6, 155.7, 172.8; [α]D25° C. 0° (c=0.53, MeOH).
2(S)-tert-Butoxycarbonylamino-4-dimethylaminobutyric acid isobutyl ester (1.6 g, 5.3 mmoles) (prepared as described in Preparative Example 30 above) was dissolved in anhydrous dichloromethane (100 mL) and the solution was cooled to 0° C. under nitrogen with stirring. Tin (II) triflate (2.21 g, 5.3 mmoles) was added in portions to the stirred solution at 0° C. The mixture was then stirred at 25° C. for 48 h. A viscous gum separated that eventually solidified. The reaction mixture was partitioned between dichloromethane and saturated aqueous sodium bicarbonate. The aqueous layer was extracted twice with dichloromethane (200 mL) and the combined extracts were dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (30×5 cm) using 3% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give unreacted 2(S)-tert-butoxycarbonylamino-4-dimethylaminobutyric acid isobutyl ester (410.5 mg, 26%) and 2(S)-(+)-amino-4-dimethylaminobutyric acid isobutyl ester (77.1 mg, 7%).
The unreacted 2(S)-tert-butoxycarbonylamino-4-dimethylaminobutyric acid isobutyl ester (410.5 mg) was taken up in 10% (v/v) concentrated sulfuric acid in dioxane (5 mL) and the mixture was stirred at 25° C. for 2 h. BioRad AG1X8 (OH−) resin was added until the pH reached 8 and the resin was then filtered off and washed with methanol. The filtrate was evaporated to dryness and the residue was chromatographed on a silica gel column (30×1.5 cm) using 5% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-(+)-amino-4-dimethylaminobutyric acid isobutyl ester (166.3 mg, 16%) (Total yield: 243.4 mg, 23%): LCMS: m/z 203.1 (MH+); HRFABMS: m/z 203.1756 (MH+). Calcd. For C10H23N2O2: m/z 203.1760; δH (CDCl3) 0.99 (6H, d, COOCH2CH(CH3)2), 1.74 (1H, m, COOCH2CH(CH3)2), 1.74 (2H, m, CHCH2CH2N(CH3)2), 2.00 (2H, m, NH2), 2.27 (6H, s, N(CH3)2), 2.41 (1H, m, CHCH2CH2N(CH3)2), 2.47 (1H, m, CHCH2CH2N(CH3)2), 3.58 (1H, m, CHCH2CH2N(CH3)2) and 3.97 ppm (2H, m, COOCH2CH(CH3)2); δC (CDCl3) CH3: 19.5, 19.5, 45.9, 45.9; CH2: 32.9, 56.7, 71.4; CH: 28.2, 53.6; C, 176.6; [α]D25° C.+2.90° (c=1.00, MeOH).
2(S)-(−)-tert-Butoxycarbonylamino-4-dimethylaminobutyric acid (prepared as described in Preparative Example 29, Step B above) may be reacted with either diazomethane, or trimethylsilyl diazomethane in a suitable inert solvent such as THF using methods well known to those skilled in the art, to give 2(S)-tert-butoxycarbonylamino-4-dimethylaminobutyric acid methyl ester.
2(S)-tert-Butoxycarbonylamino-4-dimethylaminobutyric acid methyl ester (prepared as described in Preparative Example 32 above) may be deprotected as described in Preparative Example 27, Step B to give 2(S)-amino-4-dimethylaminobutyric acid methyl ester.
2(S)-tert-Butoxycarbonylamino-4-dimethylaminobutyric acid isobutyl ester (1.5 g, 0.5 mmoles) (prepared as described in Preparative Example 30 above) was dissolved in anhydrous methanol (14 mL) and the solution was stirred and cooled to 0° C. and then saturated with anhydrous ammonia for 15 min. The vessel was sealed and stirred at 25° C. for 243 h. The reaction mixture was evaporated to dryness and the residue was chromatographed on a silica gel column (30×5 cm) using 10% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-tert-butoxycarbonylamino-4-dimethylaminobutyramide (599.5 mg, 49%): LCMS: m/z 246.1 (MH+); HRFABMS: m/z 246.1827 (MH+). Calcd. for C11H24N3O3: m/z 246.1818; 8H (CDCl3) 1.45 (9H, s, COOC(CH3)3), 1.84 (1H, m, CHCH2CH2N(CH3)2), 1.96 (1H, m, CHCH2CH2N(CH3)2), 2.23 (6H, s, N(CH3)2), 2.40 (1H, m, CHCH2CH2N(CH3)2), 2.48 (1H, m, CHCH2CH2N(CH3)2), 4.23 (1H, m, CHCH2CH2N(CH3)2), 5.23 (1H, m, NH), 6.30 (1H m, CONH2) and 7.40 ppm (1H, m, CONH2); δC (CDCl3) CH3: 28.8, 28.8, 28.8, 45.6, 45.6; CH2: 29.9, 53.9, 57.0; CH: 53.9; C, 80.2, 156.0, 174.9.
2(S)-tert-Butoxycarbonylamino-4-dimethylaminobutyramide (570 mg, 2.3 mmoles) (prepared as described in Preparative Example 34 above) was dissolved in anhydrous dichloromethane (40 mL) and the mixture was cooled to 0° C. under nitrogen with stirring. Tin (II) triflate (969.1 mg, 2.3 mmoles) was added in portions at 0° C. and the mixture stirred at 25° C. for 66 h, during which time a gummy solid separated. The reaction mixture was partitioned between dichloromethane and saturated aqueous sodium bicarbonate. The aqueous layer was extracted twice with dichloromethane (200 mL) and the latter was dried (MgSO4), filtered and evaporated to dryness. The residue was taken up in methanol (2 mL) and 10% (v/v) concentrated sulfuric acid in dioxane (10 mL) was added and the mixture was stirred at 25° C. for 3 h. The reaction mixture was diluted with methanol and BioRad AG1X8 (OH−) resin was added until the pH reached 8. The resin was filtered off and washed with methanol. The combined filtrates were evaporated to dryness to give 2(S)-amino-4-dimethylaminobutyramide (38.7 mg, 11%): LCMS: m/z 146.1 (MH+).
5-Benzyloxycarbonylamino-2(S)-tert-butoxycarbonylaminopentanoic acid (20 g, 54.6 mmoles) and 37% aqueous formaldehyde (130.1 mL, 131 mmoles) were dissolved in methanol-distilled water (1:1) (300 mL). 10% Pd—C (wet, ˜7 g) was added in portions under argon and the mixture was hydrogenated at 25° C. at 50 psi in a Parr hydrogenator for 4 days. The catalyst was filtered off through Celite® and the latter was washed with methanol-distilled water (1:1). The combined filtrates were evaporated to dryness to give 2(S)-(+)-tert-butoxycarbonylamino-4-dimethylaminopentanoic acid (14.21 g, 100%): ESMS: m/z 261.0 (MH+); Found: C, 55.15; H, 8.97: N, 10.38; C12H24N2O4 requires: C, 55.36; H, 9.29; N, 10.96; δH (CDCl3) 1.40 (9H, s, COCC(CH3)3), 1.59 (1H, m, CHCH2CH2CH2N(CH3)2) 1.77 (3H, m, CHCH2CH2CH2N(CH3)2), 2.67 (6H, s, CHCH2CH2CH2N(CH3)2), 2.77 (1H, m, CHCH2CH2CH2N(CH3)2), 2,90 (1H, m, CHCH2CH2CH2N(CH3)2), 4.06 (1H, m, CHCH2CH2CH2N(CH3)2) and 5.68 ppm (1H, d, NH); δC (CDCl3) CH3: 28.5, 28.5, 28.5, 42.7, 42.7; CH2: 21.0, 30.2, 57.9; CH: 54.5; C, 78.9, 155.5, 176.5; [α]D25° C.+23.20 (c=0.51, MeOH).
2(S)-(+)-tert-Butoxycarbonylamino-4-dimethylaminopentanoic acid (7 g, 26.9 mmoles) (prepared as described in Preparative Example 36 above), N-methylmorpholine (2.99 g, 3.25 mL, 29.6 mmoles) and isobutyl chloroformate (4.04 g, 3.84 mL, 26.9 mmoles) were dissolved in anhydrous THF (270 mL) and the mixture was stirred at −20° C. for 30 min. Conc. ammonium hydroxide (30%) (13.5 mL) was added and the mixture was stirred at 0° C. for 3 h. The mixture was evaporated to dryness and the product was chromatographed on a silica gel column (30×5 cm) using 5% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-(+)-tert-butoxycarbonylamino-4-dimethylaminopentanoic acid isobutyl ester (6.48 g, 76%): FABMS: m/z 317.2 (MH+); HRFABMS: m/z 317.2437 (MH+). Calcd. for C16H33N2O4: m/z 317.2440; δH (CDCl3) 0.93 (6H, d, COOCH2CH(CH3)2), 1.42 (9H, s, COOC(CH3)3), 1.70-1.90 (2H, m, CHCH2CH2CH2N(CH3)2), 1.94 (1H, d, COOCH2CH(CH3)2), 2.58 (6H, s, CHCH2CH2CH2N(CH3)2), 2.79 (2H, m, CHCH2CH2CH2N(CH3)2), 3.92 (2H, d, COOCH2CH(CH3)2), 4.26 (1H, m, CHCH2CH2CH2N(CH3)2) and 5.52 ppm (1H, d, NH); δC (CDCl3) CH3: 19.1, 19.1, 28.4, 28.4, 28.4, 43.7, 43.7; CH2: 21.4, 30.5, 57.8, 71.7; CH: 27.7, 52.7; C, 80.0, 155.8, 172.3; [α]D25° C.+19.90 (c=0.52, MeOH).
2(S)-(+)-tert-Butoxycarbonylamino-5-dimethylaminopentanoic acid isobutyl ester (1.0 g, 3.2 mmoles) (prepared as described in Preparative Example 37 above) was dissolved in anhydrous dichloromethane (100 mL) and the mixture was stirred at 0° C. under nitrogen. Tin (II) triflate (1.317 g, 3.2 mmoles) was added in portions at 0° C. and the mixture stirred at 25° C. for 23 h. The reaction mixture was partitioned between dichloromethane and saturated aqueous sodium bicarbonate. The dichloromethane extracts were dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on a silica gel column (30×2.5 cm) using 10% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-(+)-amino-5-dimethylaminopentanoic acid methyl ester (142.8 mg, 21%): LCMS: m/z 217.1 (MH+); HRFABMS: m/z 217.1710 (MH+). Calcd. for C11H25N2O2: m/z 217.1916; δH (CDCl3) 1.00 (6H, d, COOCH2CH(CH3)2), 1.62 (2H, m, CHCH2CH2CH2N(CH3)2), 1.77 (1H, m, CHCH2CH2CH2N(CH3)2), 1.98 (1H, m, CHCH2CH2CH2N(CH3)2), 2.28 (1H, m, COOCH2CH(CH3)2), 2.31 (6H, s, N(CH3)2), 2.40 (2H, m, CHCH2CH2CH2N(CH3)2), 3.50 (1H, m, CHCH2CH2CH2N(CH3)2) and 3.96 ppm (2H, m, COOCH2CH(CH3)2); δC (CDCl3) CH3: 18.4, 18.4, 44.2, 44.2; CH2: 23.2, 32.5, 59.3, 71.1; CH: 28.0, 54.0; C, 175.4; [α]D25° C.+0.36° (c=0.88, MeOH).
2(S)-(+)-tert-Butoxycarbonylamino-4-dimethylaminopentanoic acid (prepared as described in Preparative Example 38 above) may be reacted with either diazomethane, or trimethylsilyl diazomethane in a suitable inert solvent such as THF using methods well known to those skilled in the art, to give 2(S)-tert-butoxycarbonylamino-4-dimethylaminopentanoic acid methyl ester.
2(S)-(+)-tert-Butoxycarbonylamino-4-dimethylaminopentanoic acid methyl ester (prepared as described in Preparative Example 39 above) may be deprotected as described in Preparative Example 27, Step B to give 2(S)-amino-4-dimethylaminopentanoic acid methyl ester.
5-Benzyloxycarbonylamino-2(S)-tert-butoxycarbonylaminopentanoic acid (10 g, 27.3 mmoles), N-methylmorpholine (3.04 g, 3.3 mL, 30.0 mmoles) and isobutyl chloroformate (4.1 g, 3.89 mL, 30.0 mmoles) were dissolved in anhydrous THF (300 mL) and the mixture was stirred at −20° C. for 15 min. Conc. ammonium hydroxide (30%) (20 mL) was added and the mixture was stirred at −20 to 0° C. for 3 h. and then evaporated to dryness. The residue was chromatographed on a silica gel column (30×5 cm) using 5% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give [4(S)-(+)-tert-butoxycarbonylamino-4-carbamoylbutyl]carbamic acid benzyl ester (9.93 g, 100%): ESMS: m/z 366.2 (MH+); HRFABMS: m/z 366.2032 (MH+). Calcd. for C18H28N3O5: m/z 366.2029; δH (d6-DMSO) 1.34 (9H, s, COOC(CH3)3), 1.38 (2H, m, NHCHCH2CH2CH2NHCOO), 1.55 (1H, m, NHCHCH2CH2CH2NHCOO), 2.48 (1H, m, NHCHCH2CH2CH2NHCOO), 2.93 (2H, m, NHCHCH2CH2CH2NHCOO), 3.78 (1H, m, NHCHCH2CH2CH2NHCOO), 4.97 (2H, s, CH2C6H5), 6.68 (1H, d, NHCHCH2CH2CH2NHCOO), 6.92 (1H, d, NHCHCH2CH2CH2NHCOO), 7.20 (2H, m, CH2C6H5) and 7.32 ppm (3H, m, CH2C6H5); □C (d6-DMSO)CH3: 29.2, 29.2, 29.2; CH2: 26.1, 29.4, 65.1; CH: 53.8, 127.7, 127.7, 128.4, 128.4, 128.4; C, 77.9, 137.3, 155.3, 156.1, 174.1; [α]D25° C.+4.10 (c=0.52, MeOH).
[4(S)-(+)-tert-Butoxycarbonylamino-4-carbamoylbutyl]carbamic acid benzyl ester (6 g, 16.4 mmoles) (prepared as described in Preparative Example 41, Step A above) was dissolved in methanol (150 mL) and distilled water (50 mL) and 37% aqueous formaldehyde (3.19 mL, 39.4 mmoles) was added. 10% Pd—C (wet, ˜3.5 g) was added in portions under argon and the mixture was hydrogenated at 25° C. and 50 psi in a Parr hydrogenator for 24 h. The catalyst was filtered off through Celite® and the latter was washed with methanol-distilled water (1:1). The combined filtrates were evaporated to dryness. The residue was chromatographed on a silica gel column (30×5 cm) using 7% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give [1-carbamoyl-4(S)-(+)-dimethylaminobutyl]carbamic acid tert-butyl ester (3.33 g, 78%): FABMS: m/z 260.2 (MH+); HRFABMS: m/z 260.1982 (MH+). Calcd. for C12H26N3O3: m/z 260.1974; δH (CDCl3) 1.43 (9H, s, COOC(CH3)3), 1.58 (2H, m, NHCHCH2CH2CH2N(CH3)2), 1.80 (2H, m, NHCHCH2CH2CH2N(CH3)2), 2.22 (6H, s, NHCHCH2CH2CH2N(CH3)2), 2.31 (2H, m, NHCHCH2CH2CH2N(CH3)2), 4.08 (H, m, NHCHCH2CH2CH2N(CH3)2), 5.69 (1H, bs, NH), 6.60 (1H, bs, NH2) and 6.72 ppm (1H, bs, NH2); δC (CDCl3) CH3: 28.4, 28.4, 28.4; CH2: 23.5, 30.8, 58.9; CH: 53.8; C, 79.7, 156.2, 174.8; [α]D25° C.+2.6° (c=0.50, MeOH) and [4-dimethylamino-1(S)-(−)-(hydroxymethylcarbamoyl)butylcarbamic tert-butyl ester (466.5 mg, 10%): FABMS: m/z 290.2 (MH+); HRFABMS; m/z 290.2092 (MH+). Calcd. for C14H28N4O3: m/z 290.2080; δH (CDCl3) 1.43 (3H, s, COOC(CH3)3), 1.60 (2H, m, NHCHCH2CH2CH2N(CH3)2), 1.77 (1H, m, NHCHCH2CH2CH2N(CH3)2), 1.81 (1H, m, NHCHCH2CH2CH2N(CH3)2), 2.24 (6H, s, NHCHCH2CH2CH2N(CH3)2), 2.30 (1H, m, NHCHCH2CH2CH2N(CH3)2), 2.42 (1H, m, NHCHCH2CH2CH2N(CH3)2), 4.09 (1H, m, NHCHCH2CH2CH2N(CH3)2), 4.78 (2H, m, CONHCH2OH), 6.49 (1H, m, NHCHCH2CH2CH2N(CH3)2) and 7.92 ppm (1H, bs, CONHCH2OH); δC (CDCl3) CH3: 28.5, 28.5, 28.5; CH2: 23.2, 30.8, 58.6, 64.5; CH: 53.8; C, 79.8, 156.2, ˜174.0; [α]D25° C.−6.2° (c=0.66, MeOH).
[1-Carbamoyl-4(S)-(+)-dimethylaminobutyl]carbamic acid tert-butyl ester (3.12 g, 12.0 mmoles) (prepared as described in Preparative Example 41, Step B above) was dissolved in methanol (15 mL) and 10% (v/v) concentrated sulfuric acid in dioxane (50 mL) was added. The mixture was stirred at 25° C. for 3 h and then diluted with methanol. BioRad AG1X8 (OH−) resin was added until the pH reached 8. The resin was filtered off and washed with methanol and the combined filtrates were evaporated to dryness. The residue was chromatographed on a silica gel column (15×5 cm) using 10% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give 2(S)-(+)-amino-5-dimethylaminopentanamide (592 mg, 31%): LCMS: m/z 160.1 (MH+); HRFABMS: m/z 160.1457 (MH+). Calcd. for C7H18N3O: m/z 160.1450; δH (CDCl3) 1.70 (2H, m, CHCH2CH2CH2N(CH3)2), 1.70 (1H, m, CHCH2CH2CH2N(CH3)2), 1.83 (1H, m, CHCH2CH2CH2N(CH3)2), 2.47 (6H, s, N(CH3)2), 2.62 (2H, m, CHCH2CH2CH2N(CH3)2) and 3.72 ppm (1H, m, CHCH2CH2CH2N(CH3)2); δC (CDCl3) CH3: 43.5, 43.5; CH2: 22.8, 33.0, 58.7; CH: 54.2; C, 176.8; [α]D25° C.+4.07° (c=1.10, MeOH). The compound was found to have % Residual T @ 2 ug/mL according to scintillation proximity assay (SPA) of “D” (see description of assays below).
2-tert-Butoxycarbonyl-(S)-(+)-lysine (20 g, 81.2 mmoles) and 37% aqueous formaldehyde (19.5 mL, 19.5 mmoles) were dissolved in distilled water (300 mL). 10% Pd—C (wet, ˜7 g) was added in portions under argon and the mixture was hydrogenated at 25° C. and 50 psi in a Parr hydrogenator for 4 days The catalyst was filtered off through Celite® and the latter was washed with methanol-distilled water (1:1). The combined filtrates were evaporated to dryness to give 2(S)-(+)-tert-butoxycarbonylamino-6-dimethylaminohexanoic acid (22.53 g, 100%): ESMS: m/z 275.0 (MH+); Found: C, 55.08; H, 9.64; N, 9.69; C13H26N2O4 requires: C, 56.91; H, 9.55; N, 10.21; δH (CDCl3) 1.32 (1H, m, NHCHCH2CH2CH2CH2N(CH3)2), 1.42 (9H, S, COOC(CH3)3), 1.44 (1H, m, NHCHCHCH2CHCH2CH2N(CH3)2), 1.70 (2H, m, NHCHCH2CH2CH2CH2N(CH3)2), 1.79 (1H, m, NHCHCH2CH2CH2CH2N(CH3)2), 1.90 (1H, m, NHCHCH2CH2CH2CH2N(CH3)2), 2.68 (6H, S, NHCHCH2CH2CH2CH2N(CH3)2), 2.80 (1H, m, NHCHCH2CH2CH2CH2N(CH3)2), 2.88 (1H, m, NHCHCH2CH2CH2CH2N(CH3)2), 4.08 (1H, m, NHCHCH2CH2CH2CH2N(CH3)2) and 5.62 ppm (1H, d, NHCHCH2CH2CH2CH2N(CH3)2); δC (CDCl3) CH3: 28.3, 28.3, 28.3; CH2: 22.2, 25.0, 32.8, 57.5; CH: 54.6; C, 78.7, 155.5, 177.2; [α]D25° C.+18.50 (c=0.52, MeOH).
2(S)-(+)-tert-Butoxycarbonylamino-6-dimethylaminohexanoic acid (prepared as described in Preparative Example 43 above) may be reacted with either diazomethane, or trimethylsilyl diazomethane in a suitable inert solvent such as THF using methods well known to those skilled in the art, to give 2(S)-tert-butoxycarbonylamino-6-dimethylaminohexanoic acid methyl ester.
2(S)-tert-Butoxycarbonylamino-6-dimethylaminohexanoic acid methyl ester (prepared as described in Preparative Example 45 above) may be deprotected as described in Preparative Example 27, Step B to give 2(S)-amino-6-dimethylaminohexanoic acid methyl ester.
2(S)-(+)-tert-Butoxycarbonylamino-6-dimethylaminohexanoic acid (10 g, 36.4 mmoles) (prepared as described in Preparative Example 43 above), N-methylmorpholine (4.06 g, 4.41 mL, 40.1 mmoles) and isobutyl chloroformate (5.48 g, 5.2 mL, 40.1 mmoles) were dissolved in anhydrous THF (370 mL) and the mixture was stirred at −20° C. for 30 min. Conc. ammonium hydroxide (30%) (18.5 mL) was added and the mixture was stirred at 0° C. for 3 h. The mixture was evaporated to dryness and the product was chromatographed on a silica gel column (30×5 cm) using 7% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give [1 (S)-(+)-carbamoyl-5-dimethylaminopentyl]carbamic acid tert-butyl ester (8.81 g, 88%): FABMS: m/z 274.2 (MH+); HRFABMS: m/z 274.2129 (MH+). Calcd. for C13H28N3O3: m/z 274.2131; δH (CDCl3) 1.43 (2H, m, NHCHCH2CH2CH2CH2N(CH3)2), 1.43 (9H, s, COOC(CH3)3), 1.58 (2H, m, NHCHCH2CH2CH2CH2N(CH3)2), 1.67 (1H, m, NHCHCH2CH2CH2CH2N(CH3)2), 1.84 (1H, m, NHCHCH2CH2CH2CH2N(CH3)2), 2.32 (6H, s, NHCHCH2CH2CH2CH2N(CH3)2), 2.42 (2H, m, NHCHCH2CH2CH2CH2N(CH3)2), 4.13 (1H, m, NHCHCH2CH2CH2CH2N(CH3)2), 5.45 ppm (1H, d, NHCHCH2CH2CH2CH2N(CH3)2), 5.84 (1H, bs, CONH2) and 6.69 ppm (1H, bs, CONH2); δC (CDCl3) CH3: 28.4, 28.4, 28.4; CH2: 23.0, 26.4, 32.1, 58.9; CH: 53.9; C, 80.0, 155.9, 174.8; [α]D25° C.+2.2° (c=0.52, MeOH).
[1 (S)-(+)-Carbamoyl-5-dimethylaminopentyl]carbamic acid tert-butyl ester (prepared as described in Preparative Example 46 above) may be deprotected as described in Preparative Example 27, Step B to give 2(S)-amino-5-dimethylaminohexanoic acid amide.
PS-EDC resin (57 mg, 0.087 mmoles) was added to 96-wells of a deep well polypropylene microtiter plate followed by a MeCN/THF/DMF (5:3:2) stock solution (1 mL) of 2(S)-(+)-(2-aminomethylquinazolin-4-ylamino]-3-methylbutyric acid methyl ester (0.0291 mmoles) (prepared as described in Preparative Example 11 above) and HOBT (0.0436 mmoles). 1M stock solutions of each of the individual acids (R4COOH) (0.035 mL, 0.0348 mmoles) were added to the wells, which were then sealed and shaken at 25° C. for 20 h. The solutions were filtered through a polypropylene frit into a second microtiter plate containing PS-Isocyanate resin (3 equivalents, 0.0873 mmoles) and PS-Trisamine resin (6 equivalents, 0.175 mmoles). After the top plate was washed with MeCN (0.5 mL/well), the plate was removed, the bottom microtiter plate was sealed and then shaken at 25° C. for 16 h. The solutions were filtered through a polypropylene frit into a 96-well collection plate. The wells of the top plate were then washed with MeCN (0.5 mL/well), and the plate removed. The resultant solutions in the collection plate were transferred into vials and the solvents removed in vacuo using a SpeedVac. The resulting samples were evaluated by LCMS and those that were >70% pure are listed in the table below.
PS-DIEA resin (31 mg, 0.116 mmoles) was added to 72-wells of a deep well polypropylene microtiter plate followed by a MeCN/THF/DMF (5:3:2) stock solution (1 mL) of 2(S)-(+)-(2-aminomethylquinazolin-4-ylamino]-3-methylbutyric acid methyl ester (0.0291 mmoles) (prepared as described in Preparative Example 11 above). 1M stock solutions of each of the individual sulfonyl chlorides (R5SO2Cl) (0.043 mL, 0.043 mmoles) were added to the plate, which was then sealed and then shaken at 25° C. for 20 h. The solutions were filtered through a polypropylene frit into a second microtiter plate containing PS-Isocyanate resin (3 equivalents, 0.0873 mmoles) and PS-Trisamine resin (6 equivalents, 0.175 mmoles). After the top plate was washed with MeCN (0.5 mL/well), the plate was removed, the bottom plate was sealed and then shaken at 25° C. for 16 h. The solutions were filtered through a polypropylene frit into a 96-well collection plate. The wells of the top plate were then washed with MeCN (0.5 mL/well), and the plate removed. Then the resultant solutions in the collection plate were transferred into vials and the solvents were removed in vacuo using a SpeedVac. The resulting samples were evaluated by LCMS and those that were >70% pure are listed in the table below.
A MeCN/THF/DMF (5:3:2) stock solution of 2(S)-(−)-(2-aminomethylquinazolin-4-ylamino)-3-methylbutyric acid methyl ester (1 mL, 0.029 mmoles) (prepared as described in Preparative Example 11 above) was added to 48-wells of a deep well polypropylene microtiter plate. 1M stock solution of each of the isocyanates (R6NCO) in dichloromethane (0.06 mL, 2 equivalents, 0.058 mmoles) were added to the plate, which was then sealed and shaken at 25° C. for 20 h. The solutions were then filtered through a polypropylene frit into a second microtiter plate containing PS-Isocyanate resin (3 equivalents, 0.0873 mmoles) and PS-Trisamine resin (6 equivalents, 0.175 mmoles). After the top plate was washed with MeCN (0.5mL/well), the plate was removed, the bottom plate sealed, and then shaken at 25° C. for 16 h. Then the solutions were filtered through a polypropylene frit into a 96-well collection plate. The wells of the plate were washed with MeCN (0.5mL/well), and the top plate was removed. Then the resultant solutions in the collection plate were transferred into vials and the solvents removed in vacuo using a SpeedVac. The resulting samples were evaluated by LCMS and those that were >70% pure are listed in the table below.
PS-EDC resin (57 mg, 0.087 mmoles) was added to 96-wells of a deep well polypropylene microtiter plate followed by a MeCN/THF/DMF (5:3:2) stock solution (1 mL) of 2(S)-(+)-(2-aminomethylquinazolin-4-ylamino)-3-methylbutyramide (1 mL, 0.0233 mmoles) (prepared as described in Preparative Example 16 above) and HOBT (0.0436 mmoles). 1M stock solutions of each of the individual acids (R4COOH) (0.035 mL, 0.0348 mmoles) were added to the wells, which were then sealed and shaken at 25° C. for 20 h. The solutions were filtered through a polypropylene frit into a second microtiter plate containing PS-Isocyanate resin (3 equivalents, 0.0873 mmoles) and PS-Trisamine resin (6 equivalents, 0.175 mmoles). After the top plate was washed with MeCN (0.5 mL/well), the plate was removed, the bottom microtiter plate was sealed and then shaken at 25° C. for 16 h. The solutions were filtered through a polypropylene frit into a 96-well collection plate. The wells of the top plate were then washed with MeCN (0.5 mL/well), and the plate removed. The resultant solutions in the collection plate were transferred into vials and the solvents removed in vacuo using a SpeedVac. The resulting samples were evaluated by LCMS and those that were >70% pure are listed in the table below.
PS-DIEA resin (31 mg, 0.116 mmoles) was added to 72-wells of a deep well polypropylene microtiter plate followed by a MeCN/THF/DMF (5:3:2) stock solution (1 mL) of 2(S)-(+)-(2-aminomethylquinazolin-4-ylamino)-3-metylbutyramide (1 mL, 0.0233 mmoles) (prepared as described in Preparative Example 16 above). 1M stock solutions of each of the individual sulfonyl chlorides (R5SO2Cl) (0.043 mL, 0.043 mmoles) were added to the plate, which was then sealed and then shaken at 25° C. for 20 h. The solutions were filtered through a polypropylene frit into a second microtiter plate containing PS-Isocyanate resin (3 equivalents, 0.0873 mmoles) and PS-Trisamine resin (6 equivalents, 0.175 mmoles). After the top plate was washed with MeCN (0.5 mL/well), the plate was removed, the bottom plate was sealed and then shaken at 25° C. for 16 h. The solutions were filtered through a polypropylene frit into a 96-well collection plate. The wells of the top plate were then washed with MeCN (0.5mL/well), and the plate removed. Then the resultant solutions in the collection plate were transferred into vials and the solvents were removed in vacuo using a SpeedVac. The resulting samples were evaluated by LCMS and those that were >70% pure are listed in the table below.
A MeCN/THF/DMF (5:3:2) stock solution of 2(S)-(+)-(2-aminomethylquinazolin-4-ylamino)-3-methylbutyramide (1 mL, 0.029 mmoles) (prepared as described in Preparative Example 16 above) was added to 48-wells of a deep well polypropylene microtiter plate. 1M stock solution of each of the isocyanates (R6NCO) in dichloromethane (0.06 mL, 2 equivalents, 0.058 mmoles) were added to the plate, which was then sealed and shaken at 25° C. for 20 h. The solutions were then filtered through a polypropylene frit into a second microtiter plate containing PS-Isocyanate resin (3 equivalents, 0.0873 mmoles) and PS-Trisamine resin (6 equivalents, 0.175 mmoles). After the top plate was washed with MeCN (0.5mL/well), the plate was removed, the bottom plate sealed, and then shaken at 25° C. for 16 h. Then the solutions were filtered through a polypropylene frit into a 96-well collection plate. The wells of the plate were washed with MeCN (0.5mL/well), and the top plate was removed. Then the resultant solutions in the collection plate were transferred into vials and the solvents removed in vacuo using a SpeedVac. The resulting samples were evaluated by LCMS and those that were >70% pure are listed in the table below.
A stock solution of N,N-dimethyl-N′-[2-(methylaminomethyl)quinazolin-4-yl]propane-1,3-diamine (1 mL, 0.029 mmoles) (prepared as described in Preparative Example 20 above) in MeCN (1% AcOH) was added to 96-wells of a deepwell polypropylene microtiter plate. 1M stock solutions of each of the individual aldehydes (R1CHO) and ketones (R2COR3) in THF (0.117 mL, 0.117 mmoles) were then added, followed by an MeCN solution of tetramethylammonium-triacetoxyborohydride (18 mg, 0.0846 mmoles). The wells were then sealed and shaken at 25° C. for 20 h. To each well was then added an additional 2-equiv. of the individual aldehyde or ketone and the wells were sealed and shaken at 25° C. for 3 days. MP-TsOH resin (˜0.15 g) was then added to each well and the microtiter plate was shaken at 25° C. for 3 h. The solutions were filtered through a polypropylene frit into a 96-well collection plate. The wells on the top plate were washed three times each with dichloromethane then methanol, shaking for 5 min each time, to remove unreacted reagents, and the filtrates were discarded. Ammonia in methanol (2N, 2 mL) was then added to each well of the top microtiter plate and the latter was shaken at 25° C. for 20 min. The solutions were filtered through a polypropylene frit into a 96-well collection plate. The top microtiter plate was again shaken with ammonia in methanol (2N, 2 mL) at 25° C. for 20 min. The solutions were filtered through a polypropylene frit into a 96-well collection plate and the combined filtrates from each well, were transferred into vials and the solvents removed in vacuo using a SpeedVac. The resulting samples were evaluated by LCMS and those that were >70% pure are listed in the table below.
PS-EDC resin (57 mg, 0.087 mmoles) was added to 96-wells of a deep well polypropylene microtiter plate followed by a MeCN/THF/DMF (5:3:2) stock solution (1 mL) of N,N-dimethyl-N′-[2-(methylaminomethyl)quinazolin-4-yl]propane-1,3-diamine (1 mL, 0.0233 mmoles) (prepared as described in Preparative Example 20 above) and HOBT (0.0436 mmoles). 1M stock solutions of each of the individual acids (R4COOH) (0.035 mL, 0.0348 mmoles) were added to the wells, which were then sealed and shaken at 25° C. for 20 h. The solutions were filtered through a polypropylene frit into a second microtiter plate containing PS-Isocyanate resin (3 equivalents, 0.0873 mmoles) and PS-Trisamine resin (6 equivalents, 0.175 mmoles). After the top plate was washed with MeCN (0.5mL/well), the plate was removed, the bottom microtiter plate was sealed and then shaken at 25° C. for 16 h. The solutions were filtered through a polypropylene frit into a 96-well collection plate. The wells of the top plate were then washed with MeCN (0.5 mL/well), and the plate removed. The resultant solutions in the collection plate were transferred into vials and the solvents removed in vacuo using a SpeedVac. The resulting samples were evaluated by LCMS and those that were >70% pure are listed in the table below.
PS-DIEA resin (31 mg, 0.116 mmoles) was added to 72-wells of a deep well polypropylene microtiter plate followed by a MeCN/THF/DMF (5:3:2) stock solution (1 mL) of N,N-dimethyl-N′-[2-(methylaminomethyl)quinazolin-4-yl]propane-1,3-diamine (1 mL, 0.0233 mmoles) (prepared as described in Preparative Example 20 above). 1M stock solutions of each of the individual sulfonyl chlorides (R5SO2Cl) (0.043 mL, 0.043 mmoles) were added to the plate, which was then sealed and then shaken at 25° C. for 20 h. The solutions were filtered through a polypropylene frit into a second microtiter plate containing PS-Isocyanate resin (3 equivalents, 0.0873 mmoles) and PS-Trisamine resin (6 equivalents, 0.175 mmoles). After the top plate was washed with MeCN (0.5 mL/well), the plate was removed, the bottom plate was sealed and then shaken at 25° C. for 16 h. The solutions were filtered through a polypropylene frit into a 96-well collection plate. The wells of the top plate were then washed with MeCN (0.5mL/well), and the plate removed. Then the resultant solutions in the collection plate were transferred into vials and the solvents were removed in vacuo using a SpeedVac. The resulting samples were evaluated by LCMS and those that were >70% pure are listed in the table below.
A MeCN/THF/DMF (5:3:2) stock solution of N,N-dimethyl-N′-[2-(methylaminomethyl)quinazolin-4-yl]propane-1,3-diamine (1 mL, 0.029 mmoles) (prepared as described in Preparative Example 20 above) was added to 48-wells of a deep well polypropylene microtiter plate. 1M stock solution of each of the isocyanates (R6NCO) in dichloromethane (0.06 mL, 2 equivalents, 0.058 mmoles) were added to the plate, which was then sealed and shaken at 25° C. for 20 h. The solutions were then filtered through a polypropylene frit into a second microtiter plate containing PS-Isocyanate resin (3 equivalents, 0.0873 mmoles) and PS-Trisamine resin (6 equivalents, 0.175 mmoles). After the top plate was washed with MeCN (0.5 mL/well), the plate was removed, the bottom plate sealed, and then shaken at 25° C. for 16 h. Then the solutions were filtered through a polypropylene frit into a 96-well collection plate. The wells of the plate were washed with MeCN (0.5 mL/well), and the top plate was removed. Then the resultant solutions in the collection plate were transferred into vials and the solvents removed in vacuo using a SpeedVac. The resulting samples were evaluated by LCMS and those that were >70% pure are listed in the table below.
4-Chloro-2-(N,N-dibenzylaminomethyl)quinazoline (14.3 g, 3.85 mmoles) (prepared as described in Preparative Example 49 above) and 3-dimethylaminopropylamine (9.63 mL, 7.7 mmoles) were dissolved in 200 proof ethanol (900 mL) and the mixture was heated at 80° C. for 20 h. The solution was evaporated to dryness and the residue was chromatographed on a silica gel column (40×9 cm) using 3.5% (10% concentrated ammonium hydroxide in methanol)-dichloromethane as the eluant to give N,N-dimethyl-N′-[2-(N,N-dibenzylaminomethyl)quinazolin-4-yl]propane-1,3-diamine (16.32 g, 97%): FABMS: m/z 440.3 (MH+); HRFABMS: m/z 440.2802 (MH+). Calcd. for C28H34N5: m/z 440.2814; δH (CDCl3) 1.89 (2H, m, —NHCH2CH2CH2N(CH3)2), 2.38 (2H, m, —NHCH2CH2CH2N(CH3)2), 2.60 (2H, m, —NHCH2CH2CH2N(CH3)2), 3.84 (6H, s, (C6H5CH2)2NCH2—), 3.84 (2H, m, —NHCH2CH2CH2N(CH3)2), 7.20 (2H, m, C6H5CH2N), 7.29 (4H, m, C6H5CH2N), 7.37 (1H, ddd, H6), 7.53 (4H, d, C6H5CH2N), 7.57 (1H, dd, H5), 7.65 (1H, ddd, H7), 7.78 (1H, dd, H8) and 8.53 ppm (1H, bs, NH); δC (CDCl3) CH3: 45.6, 45.6; CH2: 24.9, 42.6, 57.6, 57.6, 59.9, 60.0; CH: 121.0, 125.2, 126.7, 126.7, 128.1, 128.1, 128.1, 128.1, 128.2, 129.1, 129.1, 129.1, 129.1; C, 114.2, 140.2, 140.2, 149.8, 159.8, 165.1.
This assay measures the growth suppression effects of small molecules in cells with mutant p53 vs. p53 null background. It uses Calcein AM to measure cellular viability. Cells (p53 null and mutant) are harvested and plated at 5000 cells per well in a 96-well tissue culture plate. The volume of cells in growth media is 100 μl. Serial dilutions (2× concentration) of compounds are then made and transferred to the plate of cells. The volume of compounds in the growth media is 100 ul. This dilution of compound with cells gives a 1× final dilution of compound (200 μL total volume). Plates are then incubated at 37° C. for 72 hours. Media is then poured off and Calcein AM is added at the appropriate concentration and the plates are incubated in the dark for 15 minutes and read for fluorescence. A letter rating corresponding to EC50 values (uM; MB468) from this assay have been assigned as follows: Compounds having EC50 values less than 2 uM have been assigned the letter “A”. Compounds having EC50 values of from 2 uM to less than 4 uM have been assigned the letter “B”. Compounds having EC50 values of from 4 uM to less than 6 uM have been assigned the letter “C”. Compounds having EC50 values of 6 uM or higher have been assigned the letter “D”.
This method assesses the ability of cells to grow in the absence of adhesion, which is a characteristic of tumorigenic cell lines. Small molecules are evaluated in this assay for their antitumor activity and the results are given in Table 3.
Human tumor DLD1 cells containing mutant p53 are suspended in growth medium containing 0.3% agarose and an indicated concentration of small molecule. The solution is overlayed onto growth medium solidified with 0.6% agarose containing the same concentration of the small molecule as the top layer. After the top layer is solidified the plates are incubated for 10-16 days at 37° C. under 5% CO2 to allow colony outgrowth. After incubation, colonies are stained by overlaying the agar with a solution of MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue; 91 mg/mL in PBS). Colonies are counted to measure growth and efficacy of the compounds of the present invention.
Most of the oncogenic mutants of the tumor suppressor protein p53 lack sequence specific DNA binding activity at physiological temperature due to conformational changes in the DNA binding domain. Small molecules and peptide which bind to the p53 DNA binding domain stabilize the conformation and restore DNA binding activity to mutant p53 protein (Science 286, 2507-2510, 1999; PNAS, 99, 937-942, 2002). Using 3H STANDARD COMPOUND (structure shown below; carbon atom with * is the atom labelled),
a radio labeled small molecule which binds to p53, and the GST-p53 DNA binding domain (aa 92-aa 312), we have developed a quantitative screening assay. The assay is based on Scintillation Proximity Assay (SPA) technology, developed by Amersham Biosciences to measure molecular interactions. Briefly, the complex of GST-p53, 3H STANDARD and Glutathione-SPA beads (Amersham Biosciences) are incubated with mixing for 1 hr at room temperature in the presence of the novel compounds to be screened. The signal is read on Microbeta. The compounds which have the ability to displace 3H STANDARD COMPOUND are selected. Such molecules will stabilize the conformation and restore DNA binding activity to mutant p53 protein.
The above assay was used to determine the ability of the compounds of this invention to directly bind to p53 core and restore DNA binding activity to mutant p53 and the results for selected compounds have been given above in various tables. Lower “% Residual total binding @ 2 ug/mL of drug” indicates superior performance.
A letter rating corresponding to % Residual total binding (T) @ 2 ug/mL of drug (i.e., compound of the present invention) from this assay have been assigned as follows: Compounds having % Residual T values of from 0% to less than 20% have been assigned the letter “A”. Compounds having % Residual T values of from 20% to less than 40% have been assigned the letter “B”. Compounds having % Residual T values of from 40% to less than 80% have been assigned the letter “C”. Compounds having % Residual T values of 80% or higher have been assigned the letter “D”. The exact % Residual T values @ 2 ug/mL for some illustrative compounds are shown below:
Unstaged Model:
In this model the therapy is started immediately after the tumor cells are inoculated.
Nude mice, 5-6 week old female, are inoculated 5×106 DLD-1 human colon adenocarcinoma cells on day 1, and randomized on day 3. The dosing of these mice is started on day 4. Groups 1 to 4, having 10 mice in each group, are dosed orally every 12 hour with vehicle, a compound of the present invention at 10 mpk, at 30 mpk, and at 50 mpk, respectively, for 31 days. All animals are carefully monitored at least daily and each tumor is measured twice a week.
Staged Model:
In this model the initiation of therapy is delayed until the tumors reach a certain volume.
Nude mice, 5-6 week old female, are inoculated with 5×106 DLD-1 human colon adenocarcinoma cells on day 1, and then randomized on day 10. The dosing of these mice is started on day 10. Groups 1 to 5, having 10 mice in each group, are dosed orally every 12 hour with no treatment, vehicle, a compound of the present invention at 10 mpk, at 30 mpk, and at 50 mpk, respectively, for 26 days. All animals are carefully monitored at least daily and each tumor is measured twice a week.
As set forth above, the compounds of the present invention potentiate the growth suppression activity of temozolomide (TMZ). This can be illustrated by showing that the compounds lower the TMZ IC50 in various cell lines. The proliferation assay that can be used for this assay is similar to that set forth above, and comprises the following general steps.
It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications that are within the spirit and scope of the invention, as defined by the appended claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 60/700,056 filed Jul. 15, 2005, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60700056 | Jul 2005 | US |
