WO2008148074A2

WO2008148074A2 - Inhibitors of mtor and methods of treatment using same

Info

Publication number: WO2008148074A2
Application number: PCT/US2008/064809
Authority: WO
Inventors: Richard Lin; Dale Drueckhammer; Jun Yong Choi; Lisa Ballou
Original assignee: Research Foundation Of State University Of New York
Priority date: 2007-05-24
Filing date: 2008-05-24
Publication date: 2008-12-04
Also published as: WO2008148074A3

Abstract

The present invention provides novel compounds that inhibit mTOR activity. Compositions, including pharmaceutical compositions, comprising compounds of the present invention are also provided. The present invention also provides methods of treatment comprising administering compositions of the present invention to a subject in need thereof.

Description

INHIBITORS OF MTOR AND METHODS OF TREATMENT USING SAME

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 60/931,858, filed May 24, 2007, which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

[0002] The invention was made with United States Government support under grant numbers DK62722 and DK59568 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Rapamycin, a bacterial macrolide, has immunosuppressant properties, and is used in kidney transplantation. Rapamycin also has antiangiogenic properties that can have dramatic antineoplastic effects, demonstrated in an animal model of metastasis. (Guba, M. et al., Nature Medicine 8, 128-135 (2002)) The mammalian target of rapamycin, commonly known as mTOR, is a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. mTOR also functions as a sensor of cellular nutrient and energy levels and redox status.

[0004] The dysregulation of the mTOR pathway is implicated as a contributing factor to various human disease processes, especially various types of cancer. Signaling through the mammalian target of rapamycin (mTOR) is hyperactivated in many human tumors, including tumors associated with tuberous sclerosis complex (TSC). Many genetic defects found in human cancers lead to hyperactivation of mTOR signaling. It is believed that certain tumor cells become dependent on this pathway because it provides a growth or survival advantage.

[0005] Therefore, drugs that inhibit mTOR have a great therapeutic potential for the treatment of cancer. Rapamycin binds to its intracellular receptor to form a complex that inhibits mTOR function. Rapamycin and its analogs, however, have two disadvantages: only some of the functions of mTOR are blocked, and Akt protein kinase, which promotes cell survival, is activated. Small molecules that can compete with ATP in the catalytic site of mTOR to inhibit the kinase-dependent functions without enhancing the cell survival functions would be very desirable to inhibit cancer growth. Thus, there remains a need for novel small molecule inhibitors of the mTOR kinase that do not have these disadvantages. These compounds may be useful for treating (TSC) as an initial treatment or in patients who are resistant to rapamycin treatment.

[0006] Tuberous sclerosis complex (TSC) is a rare genetic disease estimated to affect 1 in 6,000 individuals and most commonly manifests itself in infants and small children. (Young, J. et al, Molecular Medicine Today 4, 313-319 (1998)). TSC is characterized by the development of benign tumors called hamartomas at multiple sites in the body. Development in the brain often leads to seizures and learning and behavioral problems. The kidney, lung, and heart are also commonly affected, sometimes causing failure of these organs, while severe skin rashes are also common. Three types of brain tumors are associated with TSC: cortical tubers, which generally form on the surface of the brain; subependymal nodules, which form in the walls of the ventricles (the fluid- filled cavities of the brain); and giant-cell astrocytomas, a type of tumor that can block the flow of fluids within the brain. There is currently no cure for TSC but only symptomatic treatments. TSC is caused by mutations in either of the tumor suppressor genes tscl and tsc2. (Sparanga, S. P. et al., Current Opinion in Neurology 13, 115-119 (2000); Pan, D. et al., TRENDS in Cell Biology 14, 78-85 (2004)). These genes encode the proteins TSCl (130 kDa) and TSC2 (200 kDa), also known as hamartin and tuberin, respectively. The TSCl and TSC2 proteins form a heterodimer that negatively regulates the mammalian target of rapamycin (mTOR). (Manning, B. D. et al., Biochemical Society Transactions 31, 573-578 (2003)). The direct target of the TSC1/TSC2 complex is the small G protein Rheb (Ras homolog enriched in brain), which is a positive regulator of mTOR signaling. (Manning, B. D. et al., TRENDS in Biochemical Sciences 28, 573-576 (2003); Li, Y. et al., TRENDS in Biochemical Sciences. 29, 32-38 (2004)). The TSC1/TSC2 complex stimulates the GTPase activity of Rheb, converting it to its inactive GDP-bound state and thus inhibiting mTOR (See Fig. 2). In TSC, loss of the TSC1/TSC2 complex allows Rheb to accumulate in the active GTP-bound form, thus leading to constitutive activation of mTOR. (Young, J. et al., Molecular Medicine Today 4, 313-319 (1998); Sparanga, S. P. et al., Current Opinion in Neurology 13, 115-119 (2000); Pan, D. et al., TRENDS in Cell Biology 14, 78-85 (2004); Manning, B. D. et al., TRENDS in Biochemical Sciences 28, 573-576 (2003); Li, Y. et al., TRENDS in Biochemical Sciences 29, 32-38 (2004); Manning, B. D. et al., Biochemical Society Transactions 31, 573-578 (2003)).

[0007] mTOR is a large multidomain protein kinase that is a key component of a signaling pathway that regulates cell growth, proliferation and survival. (Crespo, J. L. et al., Microbiol MoI Biol Rev 66, 579-591 (2002); Sarbassov, D. D. et al, Science 307, 1098-1101 (2005)). The natural compound rapamycin (Fig. 1) and its analogs inhibit mTOR function without directly inhibiting its kinase activity. Experiments using these drugs in animal models have already validated mTOR as a target for the treatment of TSC. (Kenerson, H., Dundon, et al., Pediatric Research 57, 67-75; Lee, L. et al., Genes, Chromosomes and Cancer 42, 213-227 (2005); Lee, L. et al., Genes, Chromosomes and Cancer 45, 933-944 (2006)). Indeed, rapamycin is already being tested in clinical trials as a treatment for TSC. (Franz, D. N. et al., Annals of Neurology 59, 490-498 (2006).

[0008] Rapamycin (Fig. 1), a bacterial metabolite, binds to a domain of mTOR distinct from the kinase domain, resulting in inhibition of mTOR function by a mechanism that is not fully understood. Rapamycin inhibits the proliferation of many types of cells, including T cells, and is currently used as an immunosuppressant. An intact kinase domain is essential for mTOR function. The mTOR kinase domain is most closely related to the one found in phosphoinositide 3-kinases (PBKs) (see Fig. 3). (Crespo, J. L. et al., Microbiol MoI Biol Rev., 66, 579-591 (2002)). However, unlike PBKs, mTOR phosphorylates proteins, not lipids. The unusual mTOR kinase domain defines the PBK-related kinase (PIKK) family of protein kinases, which includes DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated protein kinase (ATM) and ATM- and Rad3 -related protein kinase (ATR) (see Fig. 3). Until recently, rapamycin sensitivity was the major criterion used to identify mTOR-controlled events. However, it was recently found that mTOR binds to two different regulatory subunits (Raptor and Rictor) to produce complexes with distinct signaling functions and rapamycin sensitivity. (Sarbassov, D. D. et al., Science 307, 1098- 1101 (2005); Hara, K. et al, Cell 110, 177-189 (2002); Kim, D. H. et al, Cell 110, 163-175 (2002)).

[0009] The mTOR-Raptor complex (mTORCl) phosphorylates the translation repressor 4E-BP1 and T389 in S6K (ribosomal protein S6 kinase), and is rapamycin sensitive (Fig. 2). The mTOR-Rictor complex (mTORC2) phosphorylates the protein kinase Akt at S473 and is insensitive to rapamycin. Rapamycin analogs are under clinical development as chemotherapeutic agents for a variety of cancers, and rapamycin has also been investigated for treatment of TSC. (Franz, D. N. et al., Annals of Neurology 59, 490-498 (2006); Vignot, S. et. AL, Annals of Oncology 16, 525-537 (2005)). However, as discussed above, these drugs block only some of the functions of mTOR (Sarbassov, D. D. et al., Science 307, 1098- 1101 (2005); Sarbassov, D. D. et al., Curr. Biol. 14, 1296-1302 (2004)), and they can activate Akt to promote cell survival (Tremblay, F. et al, Endocrinology 146, 1328-1337 (2005); O'Reilly, K. E. et al., Cancer Res 66, 1500-1508 (2006); Shi, Y. et al., MoI Cancer Ther 4, 1533-1540 (2005)). Recent studies indicate that Akt activity in TSCl ' or TSC2^"7" cells is abnormally low due to feedback inhibition from the hyperactivated mTORCl/S6K pathway (Yang, Q. et al., Proceedings of the National Academy of Sciences USA 103, 6811-6816 (2006); Shah, O. J. et al., Current Biology 14, 1650-1656 (2004)). Treatment of these cells with rapamycin upregulates Akt and induces resistance to the cytotoxic effect of chemotherapeutic drugs. (Shah, O. J. et al., Current Biology 14, 1650-1656 (2004)). Feedback inhibition of Akt limits the growth of TSC-related tumors (Manning, B. D. et al., Genes and Development 19, 1773-1778 (2005)), which might explain why the development of malignancy is very rare in TSC patients. (Kwiatkowski, D. J. et al., Annals of Human Genetics 67, 87-96 (2003)). Rapamycin treatment leading to inhibition of mTORCl and reactivation of Akt in these benign tumors could lead to outgrowth of more aggressive clonal populations in TSC patients.

[0010] It has been proposed that direct inhibitors of the kinase activity of mTOR will display broader anti-tumor activity than rapamycin (Bjornsti, M. A. et al., Nat Rev Cancer 4, 335-348 (2004); Guertin, D. A. et al., Trends MoI Med 11, 353-361 (2005)). Thus, the broader anti-niTOR effects of direct kinase inhibitors, which will not reactivate Akt, may be a valuable addition to the therapeutic armamentarium against TSC-related tumors.

[0011] A number of both naturally occurring and synthetic inhibitors of the PI3K and PIKK enzymes have been discovered and studied. Of these, LY294002 (Fig.l) is a structurally simple small molecule that inhibits the kinase activity of both PBKs and PIKK family members, including mTOR (Vlahos, C. J. et al., J. Biol. Chem. 269, 5241-5248 (1994)). PBKs regulate a wide range of cellular functions, including growth, glucose metabolism and motility (Katso, R., et al., Annual Review of Cellular and Developmental Biology 17, 615-675 (2001)). PIKKs regulate processes such as cell cycle progression and genome maintenance. (Abraham, R. T., DNA Repair 3, 883-887 (2004)). Therefore, use of LY294002 as a non-selective mTOR inhibitor might have undesirable toxic side effects. While the 3-D structure of mTOR has not been solved, the structures of the complexes of PBKγ with ATP, LY294002, and four other inhibitors have been reported (Walker, E. H. et al., MoI. Cell. 6, 909-919 (2000); Walker, E. H. et al., Nature 402, 313-320 (1999)). The 8- phenyl group of LY294002 binds to PBK in space occupied by the ribose moiety of ATP. The active site of PBK (and presumably the PIKK family) is more open in this position compared to more typical protein kinases, which make more extensive use of interaction with the ribose moiety in binding ATP. This may explain why LY294002 is not an inhibitor of more typical protein kinases, an important consideration in the development of selective inhibitors of mTOR.

[0012] LY294002 has been used as a lead structure for the development of isoform- selective inhibitors of PBK (Camps, M. et al, Nat. Med. 11, 936-943 (2005); Knight, Z. A. et al, Bioorg Med Chem 12, 4749-4759 (2004)), while Griffin et al. have used LY294002 as a template for the design of DNA-PK inhibitors (Griffin, R. J. et al., J Med Chem 48, 569-585 (2005); Hardcastle, I. R. et al., J Med Chem 48, 7829-7846 (2205); Leahy, J. J. et al., Med Chem Lett 14, 6083-6087 (2004)). While a large number of analogs have been reported, activity against mTOR has been reported for very few (Figure 4). Griffin et al. looked at variation of the structure 2 of Figure 4 having the 7, 8-fused benzene ring instead of the 8- phenyl group, which relative to LY294002 exhibits increased inhibition and increased selectivity toward DNA-PK vs. PI3K. (Griffin, R. J. et al., J Med Chem 48, 569-585 (2005)). A large number of substituents introduced in place of the morpholine moiety almost always resulted in greatly diminished inhibition of DNA-PK, the one exception being compound 3 (of Figure 4) having an added methyl substituent and existing as a mixture of stereoisomers due to the stereogenic center at the methyl-substituted carbon. (Griffin, R. J. et al., J Med Chem 48, 569-585 (2005)). While this substitution resulted in less than 2-fold increased inhibition of DNA-PK and mTOR relative to compound 2, it increased PI3K inhibition about 5 -fold. Compound 1 (of Figure 4) is equivalent to compound 2 (of figure 4) in geometry but has the chromenone nucleus replaced with the pyrimidoisoquinolineone structure. This modification had almost no effect on inhibition of DNA-PK and mTOR, but greatly decreased the activity against PI3Kα (see Table 2). (Griffin, R. J. et al., J Med Chem 48, 569-585 (2005)). This structure may thus be a valuable lead for the design of inhibitors of DNA-PK and/or mTOR that do not inhibit PI3Kα, though the same core structure has been employed in isoform-selective inhibitors of PI3Kβ (Jackson, S. P. et al., Nature Medicine 11, 507-514 (2005)). Substitution of the phenyl group at carbon-8 of LY294002 has also been extensively evaluated based on modification of both the original chromenone and the modified pyrimidoisoquinolineone structures, though again results for inhibition of mTOR have generally not been reported. Compound 4 (of Figure 4) and the analogous compound having oxygen in place of the sulfur atom had similar activity as LY294002 toward mTOR and PI3K, though both were greatly improved inhibitors of DNA-PK (see Table 1). (Hardcastle, I. R. et al, J Med Chem 48, 7829-7846 (2205)). Despite the extensive literature on PBK and DNA-PK inhibition, this is essentially the extent of reported mTOR inhibition data for compounds related to LY294002. LY294002 and its analogs generally do not inhibit the PIKK family members ATM and ATR at concentrations up to 100 μM. (Griffin, R. J. et al., J Med Chem 48, 569-585 (2005)).

SUMMARY OF THE INVENTION

[0013] The present invention provides novel compounds that are useful for inhibiting mTOR activity. Compounds of the present invention include those having the formula:

wherein:

A is selected from H, alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, cycloalkenyl, halo, aryl,

-(C_1-6 alkyl)-aryl, -(C_2-6 alkenyl)-aryl, -NH-C(O)-aryl, and -C(O)-NH-aryl; R¹ is selected from H, alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, cycloalkenyl, halo, aryl,

-(Ci_₆ alkyl)-aryl, and -(C_2-6 alkenyl)-aryl;

R² is selected from H, halo, amino, hydroxyl, lower alkyl, lower alkenyl, and lower alkynyl; R³ is selected from H, halo, amino, hydroxyl, lower alkyl, lower alkenyl, lower alkynyl, and cycloalkyl;

R > 4 is selected from H, halo, amino, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl,

-(CH₂VO-R⁴¹, -(CH₂)_Ω-N(R⁴²)(R⁴³), -(CH₂)_Ω-N(R⁴¹)-(CH₂),C(O)R⁴⁴, - (CH₂)_Ω-SR⁴¹, -(CH₂)_Ω-C(O)R⁴⁴, -(CH₂)_Ω-C(O)-(CH₂),OR⁴¹, -(CH₂)_Ω-C(O)-(CH₂)_δN(R⁴²)(R⁴³), -(CH₂)_ΩO-C(O)R⁴⁴, -(CH₂)_ΩS-C(O)R⁴⁴, -(CH₂)_ΩOC(O)-(CH₂),N(R⁴²)(R⁴³), - (CH₂)_Ω-As(R⁴⁴)(R⁴⁴), -(CH₂VCH=CH-(CH₂VR⁴⁰, and -(CH₂)_Ω-C(=CH₂) - (CH₂VR⁴⁰;

R⁴⁰ is selected from halo, hydroxyl, nitro, amino, CN, C(O)R⁴⁴, alkyl, cycloalkyl, aralkyl, aryl, and a heterocyclic group;

R⁴¹ is selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl and a heterocyclic group; each R⁴² and R⁴³ are independently selected from H, alkyl, cycloalkyl, cycloalkenyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic group; or R⁴² and R⁴³ may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom and may be optionally substituted with up to three substituents selected from halo, CN, NO₂, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic group; each R⁴⁴ is independently selected from H, alkyl, -OH, -O-alkyl, -O-aryl, -O-aralkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl and a heterocyclic group; a is 0 to 4; b is 0 to 4; and n is 0, 1 or 2.

[0014] Compounds of the present invention also include those having the formula:

wherein:

R¹ is selected from H, alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, cycloalkenyl, halo, aryl, -(C_1-6 alkyl)-aryl, and -(C_2-6 alkenyl)-aryl;

R² is selected from H, halo, amino, hydroxyl, lower alkyl, lower alkenyl, and lower alkynyl;

R³ is selected from H, halo, amino, hydroxyl, lower alkyl, lower alkenyl, lower alkynyl, and cycloalkyl;

R⁴ is selected from H, halo, amino, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl,

-(CH₂VO-R⁴¹, -(CH₂)_Ω-N(R⁴²)(R⁴³), -(CH₂)_Ω-N(R⁴¹)-(CH₂),C(O)R⁴⁴, -(CH₂)_Ω-SR⁴¹, -(CH₂)_Ω-C(O)R⁴⁴, -(CH₂)_Ω-C(O)-(CH₂),OR⁴¹, -(CH₂)_Ω-C(O)-(CH₂)_δN(R⁴²)(R⁴³), -(CH₂)_ΩO-C(O)R⁴⁴, -(CH₂)_ΩS-C(O)R⁴⁴, -(CH₂)_ΩOC(O)-(CH₂),N(R⁴²)(R⁴³), -(CH₂)_Ω-As(R⁴⁴)(R⁴⁴), -(CH₂VCH=CH-(CH₂VR⁴⁰, and -(CH₂)_Ω-C(=CH₂)-(CH₂)_δ-R⁴⁰;

R⁴¹ is selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl and a heterocyclic group; each R⁴² and R⁴³ are independently selected from H, alkyl, cycloalkyl, cycloalkenyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic group; or R⁴² and R⁴³ may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom and may be optionally substituted with up to three substituents selected from halo, CN, NO₂, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic group; each R⁴⁴ is independently selected from H, alkyl, -OH, -O-alkyl, -O-aryl, -O-aralkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl and a heterocyclic group; R⁵ is selected H, halo, -OH, amino, alkyl, -O-alkyl, -O-aryl, -O-aralkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, -(C_1-6 alkyl)-aryl, and -(C 2-6 alkenyl)-aryl, and a heterocyclic group; a is 0 to 4; b is 0 to 4; m is 1 or 2; and p is 0 to 3.

[0015] In certain embodiments, compounds of the invention have formula I:

wherein A is selected from:

Al A2 A3

A6

A4 A5

A7 AlO

All A12 A13 A14 and B is selected from;

Bl B2 B3

B6 B7

B9

B13

B12 or structure II:

II wherein B is selected from:

B6 B7

B9

B13

B12

[0016] Particular embodiments of the invention include compounds of formula I in which B is Bl, and A is Al, A2, A3, A4, A5, A6, A7, A8, A8, or AlO, and compounds of formula II in which B is B2, B3, B4, B5, B6, B7, B8, B9, BlO, BI l, B12, B13, or B14.

[0017] In certain embodiments, the compounds inhibit mTOR with an IC₅₀ of less than about 2μM. In certain embodiments, the compounds inhibit the kinase activity of PI3K to a lesser extent than mTOR. In certain embodiments of the invention, the compounds inhibit PI3K with an IC50 of more than about 25 μM.

[0018] The present invention also provides pharmaceutical compositions and kits comprising a compound of the present invention, and further provides methods of inhibiting mTOR activity in a cell or subject (including mammals and humans) wherein a compound of the present invention is provided to the cell or subject.

[0019] Also provided are methods of treating proliferative conditions and neoplastic diseases, including tumor-prone syndromes such tuberous sclerosis complex, and cancers.

BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1 provides the structures of rapamycin, LY294002 and a related inhibitor. LY294022 is a known inhibitor of the phosphoinositide 3-kinases (PBKs) and of the PI3K-related kinase (PIKK) family, of which mTOR is a member. (Vlahos, C. J. et al, J. Biol. Chem. 269, 5241-5248 (1994); Abraham, R. T., DNA Repair. 3, 883-887 (2004); Brunn, G. J., EMBO Journal 15, 5256-5267 (1996)). Compound 1 of figure 1 has been developed as an inhibitor of the related DNA-dependent protein kinase (DNA-PK) (Griffin et al 2005).

[0021] Figure 2 provides a schematic description of TSC-related cell signaling pathways. [0022] Figure 3 provides an alignment of kinase domains of PBKs and PIKKs. The shading points out some of the most highly conserved amino acids among the six sequences. The diamonds point out two positions in which sequence differences are being exploited for selective inhibition of mTOR.

[0023] Figure 4 provides the structure and IC₅₀ values (μM) for LY294002 and analogs for which mTOR inhibition data have been reported.

[0024] Figure 5 provides active site residues (tube structures) and LY294002 (space filling model) in the kinase domains of PBKγ (left) and mTOR (right). The mTOR structure was derived from homology modeling based on the crystal structure of PBKγ using the MOE software package.

[0025] Figure 6 shows carbon-8 substituents of some compounds of structure I of the present invention. Also shown are approximate IC₅₀ values for mTOR (upper value) and PBK (lower, α or β isoform as indicated). When only one value is shown, it is for mTOR.

[0026] Figure 7 provides the results of in vitro kinase assays. Figure 7A shows an autoradiograph where immunoprecipitated mTOR was assayed with increasing concentrations of compound 1 of Figure 1 in μM. Figure 7B shows the results of a study where PBKα (represented by circles) and PBKβ (represented by squares) was purified from baculovirus-infected insect cells and assayed in the presence of compound 1 of Figure 1 (closed) or LY294002 (open).

[0027] Figure 8 provides the results of in vivo assays. Figure 8A shows the results of a study where serum- starved Ratl cells were pretreated with vehicle (Veh), 10 nM rapamycin (Rap), 5 μM compound 1 of Figure 1 or 5 μM LY294002 for 20 min. and then treated with or without 50 ng/ml PDGF (platelet-derived growth factor) for 10 min. Figure 8B shows the results of a study where MCF7 cells growing in serum-containing medium were treated with vehicle, 10 nM rapamycin or 25 μM of compound 1 for 24 h. Cell lysates were analyzed by Western blotting using the indicated antibodies, including actin to show equal loading of proteins in each lane. Figure 8C shows the results of a study where MCF7 cells were treated with increasing concentrations of compound 1 of figure 1 for 4 days and cell growth was measured by MTT assays.

[0028] Figure 9 provides compounds of the present invention with a quinoline or isoquinoline ring at C-8. [0029] Figure 10 provides compounds of the present invention — acylthio and bromo analogs as C2243 -modifying inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides novel compounds that are useful in inhibiting mTOR activity. Guided by computation homology modeling of the mTOR kinase domain, a series of compounds based on a pyrimidoisoquinolineone ring system designed to discriminate between the active sites of mTOR and related kinases were synthesized and tested. The compounds of the present invention were designed based primarily on the crystal structure of the PI3Kγ-LY294002 complex (Walker, E. H. et al, MoI. Cell. 6, 909-919 (2000); Walker, E. H. et al., Nature 402, 313-320 (1999)). Using this structure as a template, 3-D structures for the corresponding complexes with the kinase domains of PI3Kα, mTOR and DNA-PK have been developed by homology modeling using the Molecular Operating Environment program (MOE, Chemical Computing Group, Montreal, Canada). Fig. 3 shows an alignment of a portion of the active site sequences of selected PI3K and PIKK family members. Views of the PI3Kγ active site structure alongside the mTOR homology model are shown in Fig. 5. The results revealed two interesting differences in active site residues between these kinases (indicated by the diamonds in Figure 3).

[0031] First, the side chain indole of W812 in PI3Kγ is very close to the carbon-8 phenyl group of LY294002 in the crystal structure. The equivalent residue is also W in the other PBKs but is K in mTOR (K2171) and ATR, R in DNA-PK, and I in ATM. The 3-D models indicate that the active sites of mTOR and DNA-PK have additional space in this area that can accommodate expansion of the 8-phenyl ring of LY294002 due to the less bulky K and R side chains. The W2239 side chain of mTOR, equivalent to 1881 in PBKγ and present as W in all of the PIKKs, extends into some of this space but still leaves room for expansion of the inhibitor (Figure 5).

[0032] Second, the methyl side chain of A885 in PBKγ is about 5A away from one of the methylene carbons attached to oxygen in the morpholine moiety of LY294002 (Fig. 5). The equivalent amino acid is S or T in most of the other members of this family while mTOR is the only member that has C in this position. C2243 in mTOR thus provides a potential site for covalent modification by a group on the morpholine moiety of the inhibitor that reacts with the thiol, which thus imparts additional selectivity for mTOR. [0033] The compounds of the present invention exploit the structural differences described above, based on the LY294002 structure but with the arrangement of heteroatoms of the pyrimidoisoquinolineone ring system of 1 (Figure 1).

[0034] In certain embodiments, the compounds of the present invention have the structure:

wherein:

-(Ci_6 alkyl)-aryl, and -(C_2-6 alkenyl)-aryl;

-(CH₂VO-R⁴¹, -(CH₂)_Ω-N(R⁴²)(R⁴³), -(CH₂)_Ω-N(R⁴¹)-(CH₂),C(O)R⁴⁴, -(CH₂)_Ω-SR⁴¹, -(CH₂)_Ω-C(O)R⁴⁴, -(CH₂)_Ω-C(O)-(CH₂),OR⁴¹, -(CH₂)_Ω-C(O)-(CH₂)_δN(R⁴²)(R⁴³), -(CH₂)_ΩO-C(O)R⁴⁴, -(CH₂)_ΩS-C(O)R⁴⁴, -(CH₂)_ΩOC(O)-(CH₂),N(R⁴²)(R⁴³), -(CH₂)_Ω-As(R⁴⁴)(R⁴⁴), -(CH₂VCH=CH-(CH₂VR⁴⁰, and -(CH₂)_Ω-C(=CH₂)-(CH₂),-R⁴⁰;

[0035] In certain preferred embodiments for compounds of the formula IA, R² is selected from H, halo, amino, hydroxyl, methyl and trifluoromethyl. In particularly preferred embodiments R² is H.

[0036] In certain preferred embodiments for compounds of the formula IA, R³ is selected from H, halo, amino, hydroxyl, methyl and trifluoromethyl. In particularly preferred embodiments R³ is H.

[0037] In certain preferred embodiments for compounds of the formula IA, A is selected from aryl, -(C_1-6 alkyl)-aryl, -(C_2-6 alkenyl)-aryl.

[0038] In certain preferred embodiments for compounds of the formula IA, R¹ is selected from H, halo, and alkyl. In particularly preferred embodiments R¹ is H.

[0039] In certain preferred embodiments for compounds of the formula IA, R⁴ is selected from halo, amino, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, -(CH₂)O-O-R⁴¹, -(CH₂)_Ω-N(R⁴²)(R⁴³), -(CH₂)_Ω-N(R⁴¹)-(CH₂),C(O)R⁴⁴, -(CH₂)_Ω-SR⁴¹, -(CH₂)_Ω-C(O)R⁴⁴, -(CH₂)_Ω-C(O)-(CH₂),OR⁴¹, -(CH₂)_Ω-C(O)-(CH₂),N(R⁴²)(R⁴³), -(CH₂)_ΩO-C(O)R⁴⁴, -(CH₂)_ΩS-C(O)R⁴⁴, -(CH₂)_ΩOC(O)-(CH₂)_δN(R⁴²)(R⁴³), -(CH₂)_Ω-CH=CH-(CH₂)_δ-R⁴⁰, and -(CH₂)_Ω-C(=CH₂)-(CH₂),-R⁴⁰.

[0040] In other embodiments, the compounds of the present invention have the structure:

wherein:

R¹ is selected from H, alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, cycloalkenyl, halo, aryl,

-(C_1-6 alkyl)-aryl, and -(C₂_6 alkenyl)-aryl; R² is selected from H, halo, amino, hydroxyl, lower alkyl, lower alkenyl, and lower alkynyl; R³ is selected from H, halo, amino, hydroxyl, lower alkyl, lower alkenyl, lower alkynyl, and cycloalkyl;

-(CH₂VO-R⁴¹, -(CH₂VN(R⁴²XR⁴³), -(CH₂)_Ω-N(R⁴¹)-(CH₂),C(O)R⁴⁴, -(CH₂)_Ω-SR⁴¹, -(CH₂)_Ω-C(O)R⁴⁴, -(CH₂)_Ω-C(O)-(CH₂),OR⁴¹, -(CH₂)_Ω-C(O)-(CH₂),N(R⁴²)(R⁴³), -(CH₂)_ΩO-C(O)R⁴⁴, -(CH₂)_ΩS-C(O)R⁴⁴, -(CH₂)_ΩOC(O)-(CH₂),N(R⁴²)(R⁴³), -(CH₂)_Ω-As(R⁴⁴)(R⁴⁴), -(CH₂)_Ω-CH=CH-(CH₂)_δ-R⁴⁰, and -(CH₂)_Ω-C(=CH₂)-(CH₂)_δ-R⁴⁰;

R⁴¹ is selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl and a heterocyclic group; each R⁴² and R⁴³ are independently selected from H, alkyl, cycloalkyl, cycloalkenyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic group; or R⁴² and R⁴³ may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom and may be optionally substituted with up to three substituents selected from halo, CN, NO₂, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic group; each R⁴⁴ is independently selected from H, alkyl, -OH, -O-alkyl, -O-aryl, -O-aralkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl and a heterocyclic group;

R⁵ is selected H, halo, -OH, amino, alkyl, -O-alkyl, -O-aryl, -O-aralkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, -(C_1-6 alkyl)-aryl, and -(C₂_₆ alkenyl)-aryl, and a heterocyclic group; a is O to 4; b is O to 4; m is 1 or 2; and

/? is O to 3.

[0041] In certain preferred embodiments for compounds of the formula HA, R² is selected from H, halo, amino, hydroxyl, methyl and trifluoromethyl. In particularly preferred embodiments R² is H.

[0042] In certain preferred embodiments for compounds of the formula HA, R is selected from H, halo, amino, hydroxyl, methyl and trifluoromethyl. In particularly preferred embodiments R³ is H. [0043] In certain preferred embodiments for compounds of the formula HA, R⁴ is selected from halo, amino, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, -(CH₂)_Ω-O-R⁴¹, -(CH₂)_Ω-N(R⁴²)(R⁴³), -(CH₂)_Ω-N(R⁴¹)-(CH₂),C(O)R⁴⁴, -(CH₂)_Ω-SR⁴¹, -(CH₂)_Ω-C(O)R⁴⁴, -(CH₂)_Ω-C(O)-(CH₂),OR⁴¹, -(CH₂)_Ω-C(O)-(CH₂),N(R⁴²)(R⁴³), -(CH₂)_ΩO-C(O)R⁴⁴, -(CH₂)_ΩS-C(O)R⁴⁴, -(CH₂)_ΩOC(O)-(CH₂)_δN(R⁴²)(R⁴³), -(CH₂)_Ω-CH=CH-(CH₂)_δ-R⁴⁰, and -(CH₂)_Ω-C(=CH₂)-(CH₂),-R⁴⁰.

[0044] The term "halo" or "halogen" as used herein includes fluorine, chlorine, bromine and iodine.

[0045] The term "alkyl" as used herein contemplates substituted or unsubstituted, straight and branched chain alkyl radicals containing from one to eight carbon atoms. The term "lower alkyl" as used herein contemplates both straight and branched chain alkyl radicals containing from one to six carbon atoms and includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like. The alkyl group may be optionally substituted with one or more substituents selected from halo, oxo, CN, NO₂, CO₂R, C(O)R, -O-R, -N(R)(R), -N(R)C(O)R, -N(R)SO₂R, -SR, -C(O)N(R)(R), -OC(O)R, -SC(O)R, -OC(O)N(R)(R), SO₂, -SOR, -SO₃R, and -SO₂N(R)(R).

[0046] The term "alkenyl" as used herein contemplates substituted or unsubstituted, straight and branched chain alkene radicals containing from two to eight carbon atoms. The term "lower alkenyl" as used herein contemplates alkenyl radicals containing from two to six carbon atoms. The alkenyl group may be optionally substituted with one or more substituents selected from halo, oxo, CN, NO₂, CO₂R, C(O)R, -O-R, -N(R)(R), -N(R)C(O)R, -N(R)SO₂R, -SR, -C(O)N(R)(R), -OC(O)R, -SC(O)R, -OC(O)N(R)(R), SO₂, -SOR, -SO₃R, and -SO₂N(R)(R).

[0047] The term "alkynyl" as used herein contemplates substituted or unsubstituted, straight and branched carbon chain containing from two to 8 carbon atoms and having at least one carbon-carbon triple bond. The term alkynyl includes, for example ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 3-methyl-l-butynyl, and the like. The term "lower alkynyl" as used herein contemplates alkenyl radicals containing from two to six carbon atoms. The alkynyl group may be optionally substituted with one or more substituents selected from halo, oxo, CN, NO₂, CO₂R, C(O)R, -O-R, -N(R)(R), -N(R)C(O)R, -N(R)SO₂R, -SR, -C(O)N(R)(R), -OC(O)R, -SC(O)R, -OC(O)N(R)(R), SO₂, -SOR, -SO₃R, and -SO₂N(R)(R). [0048] The term "cycloalkyl" as used herein contemplates substituted or unsubstituted cyclic alkyl radicals containing form 3 to 7 carbon atoms and includes cyclopropyl, cyclopentyl, cyclohexyl, and the like. A cycloalkyl group may be optionally substituted with one or more substituents selected from halo, oxo, CN, NO₂, CO₂R, C(O)R, -O-R, -N(R)(R), -N(R)C(O)R, -N(R)SO₂R, -SR, -C(O)N(R)(R), -OC(O)R, -SC(O)R, -OC(O)N(R)(R), SO₂, -SOR, -SO₃R, and -SO₂N(R)(R).

[0049] The term "cycloalkenyl" as used herein contemplates substituted or unsubstituted cyclic alkenyl radicals containing form 5 to 7 carbon atoms in which has a double bond between two of the ring carbons and includes cyclopentenyl, cyclohexenyl, and the like. A cycloalkenyl group may be optionally substituted with one or more substituents selected from halo, oxo, CN, NO₂, CO₂R, C(O)R, -O-R, -N(R)(R), -N(R)C(O)R, -N(R)SO₂R, -SR, -C(O)N(R)(R), -OC(O)R, -SC(O)R, -OC(O)N(R)(R), SO₂, -SOR, -SO₃R, and -SO₂N(R)(R).

[0050] The terms "aryl" as used herein contemplates substituted or unsubstituted single-ring aromatic groups (for example, phenyl, pyridyl, pyrazole, etc.) and fused polycyclic ring systems (naphthyl, quinoline, dibenzothiophene, etc.), and unfused polycyclic ring systems (biphenyl, bipyridine, phenyl-pyridine, etc.). The polycyclic rings may have two or more rings in which two atoms are common to two adjoining rings (the rings are "fused") wherein at least one of the rings is aromatic, e.g., the other rings can be cycloalkyls, cycloalkenyls, aryl, heterocycles and/or heteroaryls. The aryl group may be optionally substituted with one or more substituents selected from halo, oxo, CN, NO₂, CO₂R, C(O)R, -O-R, -N(R)(R), -N(R)C(O)R, -N(R)SO₂R, -SR, -C(O)N(R)(R), -OC(O)R, -SC(O)R, -OC(O)N(R)(R), SO₂, -SOR, -SO₃R, and -SO₂N(R)(R).

[0051] The term "heterocyclic group" or "heterocyclic ring" as used herein contemplates substituted or unsubstituted aromatic and non-aromatic cyclic radicals having at least one heteroatom as a ring member. Preferred heterocyclic groups are those containing 5 or 6 ring atoms which includes at least one hetero atom, and includes cyclic amines such as morpholino, piperidino, pyrrolidino, and the like, and cyclic ethers, such as tetrahydrofuran, tetrahydropyran, and the like. Aromatic heterocyclic groups, also termed "heteroaryl" groups contemplates single-ring hetero-aromatic groups that may include from one to three heteroatoms, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like. The term heterocyclic group also includes polycyclic ring systems having two or more rings in which two atoms are common to two adjoining rings (the rings are "fused") wherein at least one of the rings is a heterocycle, e.g., the other ring(s) can be cycloalkyls, cycloalkenyls, aryl, heterocycles and/or heteroaryls. Examples of polycyclic heteroaromatic systems include quinoline, isoquinoline, tetrahydroisoquinoline, quinoxaline, quinaxoline, benzimidazole, benzofuran, purine, imidazopyridine, benzotriazole, and the like. A heterocyclic group may be optionally substituted with one or more substituents selected from halo, oxo, CN, NO₂, CO₂R, C(O)R, -0-R, -N(R)(R), -N(R)C(O)R, -N(R)SO₂R, -SR, -C(O)N(R)(R), -OC(O)R, -SC(O)R, -OC(O)N(R)(R), SO₂, -SOR, -SO₃R, and -SO₂N(R)(R).

[0052] The term "aralkyl" as used herein contemplates a lower alkyl group which has as a substituent an aryl group,

[0053] With respect to the above definitions, each R is independently selected from H, Ci_₆ alkyl, C₃_₆ cycloalkyl, C₂_₆ alkenyl, aryl, and aralkyl.

[0054] The term "heteroatom", particularly as a ring heteroatom, refers to N, O, and S.

[0055] Preferred compounds of the present invention have structure I:

wherein A is selected from:

Al A2 A3

A6

A4 A5

All A12 A13 A14

and B is selected from

Bl

B3

B6 B7

B8 B9

B13

B12

B14 B 15

or structure II:

II wherein B is selected from B2-B18.

[0056] For compounds of structure I all combinations of A and B are within the scope of the invention. For compounds of structure II, B2-B18 are within the scope of the invention.

[0057] In one embodiment, the compound is structure I, wherein A is Al and B is Bl . [0058] In one embodiment, the compound is structure I, wherein A is A2 and B is Bl .

[0059] In one embodiment, the compound is structure I, wherein A is A3 and B is Bl .

[0060] In one embodiment, the compound is structure I, wherein A is A4 and B is Bl .

[0061] In one embodiment, the compound is structure I, wherein A is A5 and B is Bl .

[0062] In one embodiment, the compound is structure I, wherein A is A6 and B is Bl .

[0063] In one embodiment, the compound is structure I, wherein A is A7 and B is Bl .

[0064] In one embodiment, the compound is structure I, wherein A is A8 and B is Bl .

[0065] In one embodiment, the compound is structure I, wherein A is A9 and B is Bl .

[0066] In one embodiment, the compound is structure I, wherein A is AlO and B is Bl.

[0067] In one embodiment, the compound is structure II and B is B2.

[0068] In one embodiment, the compound is structure II and B is B3.

[0069] In one embodiment, the compound is structure II and B is B4.

[0070] In one embodiment, the compound is structure II and B is B5.

[0071] In one embodiment, the compound is structure II and B is B6.

[0072] In one embodiment, the compound is structure II and B is B7.

[0073] In one embodiment, the compound is structure II and B is B8.

[0074] In one embodiment, the compound is structure II and B is B9.

[0075] In one embodiment, the compound is structure II and B is BlO.

[0076] In one embodiment, the compound is structure II and B is Bl 1.

[0077] In one embodiment, the compound is structure II and B is B 12.

[0078] In one embodiment, the compound is structure II and B is B13.

[0079] In one embodiment, the compound is structure II and B is B 14.

[0080] In one embodiment, the compound is structure II and B is B15.

[0081] In one embodiment, the compound is structure II and B is B 16.

[0082] In one embodiment, the compound is structure II and B is B 17. [0083] In one embodiment, the compound is structure II and B is B 18.

[0084] Compounds of the invention are mTOR inhibitors. In certain embodiments, the IC50 for mTOR inhibition is less than about 10 μM, or less than about 5 μM, or less that about 2 μM. In certain embodiments, a compound of the invention is a more effective inhibitor of mTOR than of a related kinase. Thus, according to the invention, certain mTOR inhibitors have an IC50 for mTOR that is at least about 2 fold, or at least about 5 fold, or at least about 10 fold lower than the IC50 for PBKα. In an embodiment of the invention, the IC₅₀ for PBKα is greater than about 10 μM or greater than about 25 μM. Preferred compounds of the present invention include, but are not limited to, compounds where the structure is structure I and wherein A is A2 or A6. Another preferred compound is a compound having structure II wherein B is B9.

[0085] The compounds of formula I wherein A is an aromatic group (A1-A5 and Al 1 -Al 4) and B is Bl can be made from compound 4 by the Suzuki coupling reaction with an arylboronic acid as depicted below:

I, wherein A is aryl wherein Ar is A1-A5 or Al 1-A14 (See, Suzuki, A. Pure Appl. Chem. (1991) 63, 419-422; Miyaura, N.; Suzuki, A. Chem. Rev. (1995) 95, 2457-2483; and Suzuki, A. J. Organometallic Chem. (1999) 576, 147-168): "One Pot Biaryl Synthesis via in situ Boronate Formation" Giroux, A; Han, Y.; Prasit, P. Tetrahedron Lett. 1997, 38, 3841-3844; "New Air-Stable Catalysts for General and Efficient Suzuki-Miyaura Cross-Coupling reactions of Heteroaryl Chlorides" Guram, A.S.; King, A.O.; Allen, J.G.; Wang, X.; Schenkel, L.B.; Chan, J.; Bunel, E.E.; Gaul, M. M.; Larsen, R. D.; Martinelli, M. J.; Reider, P. J. Org. Lett; 2006; 8(9); 1787- 1789.)

[0086] Alternatively, the iodide of compound 4 can be converted to a boronic acid functional group that can then be coupled with an aryl halide (aryl = A1-A5 or Al 1 -Al 4) using the Suzuki coupling reaction to provide compounds of formula I, wherein A is aryl. (See "Organoboranes 31. A Simple Preparation of Boronic Esters from Organo-Lithium Reagents and Selected Trialkoxyboranes" Brown, H. C; Cole, T.E. Organometallics 1983, 2, 1316-1319: "Palladium (O)-Catalyzed Cross-Coupling Reaction of Alkoxydiboron with Halorenes: A Direct Procedure for Arylboronic Esters" Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508-7510.)

[0087] The compounds of formula I wherein A is an aryl alkene group (A6-A9) can be made from compound 4 by the Heck reaction with an aryl alkene as depicted below:

DMF

I, wherein A is aryl alkene wherein Ar-CH=CH₂ is A6-A9 (See, N.T.S. Phan, et. al, (2006) On the Nature of the Active Species in Palladium Catalyzed-Mizoroki-Heck and Mizoroki-Miyaura Couplings, Homogeneous or Heterogeneous Catalysis, A critical Review, Advanced Synthesis and Catalysis, 348, 609-679; Heck, R. F. Org. React. (1982), 27, 345-390; A. de Meijere, F. E. Meyer, Jr., Angew. Chem. Int. Ed. Eng. (1994), 33(23-24), 2379-2411; and I. P. Belestskaya and A.V. Cheprakov, Chem. Rev. (2000), 100, 3009-3066).

[0088] The compound of formula I wherein A is a benzamido group (i.e., AlO) is obtained by a Pd-catalyzed coupling of benzamide with compound 4 (See, J.J. Yin and S. L. Buchwald, (2000), Palladium Catalyzed Intermolecular Coupling of Aryl Halides and Amides, Organic Letters, 2, 1101-1104).

[0089] Compound 4 can be prepared by the following synthetic scheme:

Scheme 1

(See, R.J. Griffin et al., (2205) Selective Benzopyranone and Pyrimido[2,l-a]isoquinolin-4- one Inhibitors of DNA-dependent Protein Kinase: Synthesis, Structure- Activity Studies, and Radiosensitization of a Human Tumor Cell Line In Vitro, J. Med. Chem., 48, 569-585 and L. Estel et al., (1988) Metalation/SRNl Coupling in Heterocyclic Synthesis. A convenient Methodology for Ring Functionalization, J. Org. Chem. 53, 2740-2744).

[0090] Compounds of structure I, wherein B is a substituted morpholino group can be made by a similar reaction wherein compound 3 is reacted with a compound of formula 5 to provide a compound of formula 6 as depicted below:

[0091] The A group is then introduced into compound 6 by either the Suzuki coupling reaction or the Heck reaction as described above. After the A group is introduced the hydroxyl of the resulting compound can be converted into a leaving group (for example, a tosylate or mesylate, by reaction with toluenesulfonyl chloride or methanesulfonyl chloride in the presence of diethylamine, respectively) to provide a compound of formula 7.

wherein X is a leaving group.

[0092] Although, compound 5 is depicted above as the racemic mixture, the individual isomers of compound 5, i.e., the (R) isomer substantially free of the (S) isomer and (S) isomer substantially free of the (R) isomer are also within the scope of the invention. Accordingly, compounds of structure I and II wherein B is an individual isomer of B2-B5, B9, or B 12 are also within the scope of invention. Individual isomers of compound 5 will be prepared as described in the literature. (See, Berg, S., Larsson, L. G., Renyi, L., Ross, S.B., Thorberg, S.O. and Thorell-Scantesson, G. "(R)-(+)-2[[[3-(Morpholinomethyl)-2H-chromen- 8-yl]oxy]methyl] morpholine methanesulfonate: a new selective rat 5-hydrocytryptamine IB receptor antagonist." JMed Chem, 1998, 41, 1934-1942; "Asymmetric Synthesis of (+)-(S,S)- Rebocetine via a New (5)-2-(Hydroxymethyl)morpholine Preparation" Eric Brenner, Ronald M. Baldwin, and Gilles Tamagnan, Org Lett. 2005, 7, 937-939.)

[0093] Compound 7 can be reacted with Cl^" or Br^" (for example, NaCl or NaBr) to provide the compound of structure I wherein B is B2 or B3, respectively. Compound 7 can also be reacted with CH₃-C(O)SH in the presence of NaH in dimethyl formamide to provide the compound of structure I wherein B is B4. Hydrolysis of the compounds of structure I wherein B is B4, for example, with sodium hydroxide in methanol, provides the compound of structure I wherein B is B9. Reaction of the compound of structure I wherein B is B9 with an acid chloride of formula R-C(O)Cl, wherein R is an alkyl group, provides a thioester. For example if R in R-C(O)Cl is -CH(CH₃ )₂ the resulting compound is the compound of structure I wherein B is B5.

[0094] Oxidation of the hydroxyl group of compound 6 under appropriate mild conditions should provide the compound of formula I wherein B is B 12. (For a review of oxidations, see, for example F.A. Carey and R.J. Sundberg, Advanced Organic Chemistry, 4^th ed. Part B, Chapter 12 (2000)). Swern oxidation (i.e., oxalyl chloride, dimethyl sulfoxide, and triethyl amine) or TEMPO oxidation (i.e., TCIA (trichloroisocyanuric acid) and NaHCO₃) of compound 6, however, are not effective. (See, Oxidation of alcohols by "activated" dimethyl sulfoxide. A prepartive, steric and mechanistic study. Kanji Omura and Daniel Swern, Tetrahedron 1978, 34, 1651-1660; Lidia De Luca, Giampaolo Giacomelli, and Andrea Porcheddu, Org. Lett. 2001, 3, 3041-3043.)

[0095] Compounds of structure I wherein B is B6-B8 are obtained by reacting compound 3 with a compound of formula 9:

which is prepared from the known ester 8 as depicted below:

LiAlH,

(See, D.J. Blythin et al, (1996) Substituted Morpholine-2S-Acetic Acid Derivatives: SCH 50911 and Related Compounds as Novel GABAB Antagonists, Bioorganic and Medicinal Chemistry Letters 6, 1529-1534).

[0096] Compound 9 is then coupled with compound 3 to provide compound 10.

[0097] The A group is then introduced into compound 10 by either the Suzuki coupling reaction or the Heck reaction to provide compound 11 :

[0098] The hydroxyl of compound 11 can be functionalized as described above. For example, the hydroxyl of compound 11 can be converted into a leaving group and then converted into -Cl, -Br, or -SH, i.e., compounds of structure I wherein B is B6, B7, or B 13, using the methods described above. The compounds of structure I wherein B is -SC(O)CH3, or -SC(O)CH(CHs)₂, i.e., compounds of structure I wherein B is BlO or BI l, respectively, are also prepared from compound 11 using the methods described above. Oxidation of the hydroxyl of compound 11 (Swern oxidation (i.e., oxalyl chloride, dimethyl sulfoxide, and triethyl amine) or TEMPO oxidation (i.e., TCIA and NaHCOs) provides the compound structure I wherein B is B8.

[0099] Although compound 9 is depicted above as the racemic mixture, the individual isomers of compound 9, i.e., the (R) isomer substantially free of the (S) isomer and (S) isomer substantially free of the (R) isomer are also within the scope of the invention. Accordingly, compounds of structures I and II wherein B is an individual isomer of B6-B8 or B 10-Bl 3 are also within the scope of invention.

[0100] Compounds of structure II are prepared by reacting a compound of formula 12 with a compound of formula 5 or 9 and then functionalizing the hydroxyl of the resulting compound as described above. Compound 12 is a known compound:

(See "Selective Benzopyranone and Pyrimido[2,l-α]isoquinolin-4-one Inhibitors of DNA- Dependent Protein Kinase: Synthesis, Structure- Activity Studies, and Raiosensitization of a Human Tumor Cell Line in Vitro" Roger J. Griffin, Gabriele Fontana, Bernard T. Golding, Sophie Guiard, Ian R. Hardcastle, Justin J. J. Leahy, Niall Martin, Caroline Richardson, Laurent Rigoreau, Martin Stockley, and Graeme C. M. Smith, J Med Chem 2005, 48, 569- 585.)

[0101] The present invention further provides methods of inhibiting mTOR activity in a cell or subject wherein a compound of the present invention is provided to the cell or subject. The subject can be a mammal such as a human. Preferable compounds inhibit mTOR activity, including both mTOR-Raptor (mTORCl) and mTOR-Rictor (mTORC2). More preferred compounds also are selective inhibitors of mTOR, that inhibit the kinase activity of PBK and/or other PIKK family members to a lesser extent than mTOR. Preferred compounds do not reactivate or activate the protein kinase Akt. Inhibit means reduce, decrease or completely eliminate activity.

[0102] The present invention also provides pharmaceutical compositions comprising a compound of the present invention and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is one that does not cause an adverse physical reaction upon administration and one in which compounds of the present invention are sufficiently soluble and retain their activity to deliver a therapeutically effective amount of the compound. The therapeutically effective amount and method of administration of compounds of the invention may vary based on the individual patient, the indication being treated and other criteria evident to one of ordinary skill in the art. A therapeutically effective amount of a compound of the invention is one sufficient to inhibit mTOR activity without causing significant adverse side effects. The route(s) of administration useful in a particular application are apparent to one of ordinary skill in the art.

[0103] A salt of a compound of this invention is formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group. According to another embodiment, the compound is a pharmaceutically acceptable acid addition salt.

[0104] The term "pharmaceutically acceptable," as used herein, refers to a component that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. A "pharmaceutically acceptable salt" means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention. A "pharmaceutically acceptable counterion" is an ionic portion of a salt that is not toxic when released from the salt upon administration to a recipient.

[0105] Acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para¬

ng- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephathalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β- hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, naphthalene-2- sulfonate, mandelate and other salts. In one embodiment, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.

[0106] As used herein, the term "hydrate" means a compound which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0107] As used herein, the term "solvate" means a compound which further includes a stoichiometric or non-stoichiometric amount of solvent such as water, acetone, ethanol, methanol, dichloromethane, 2-propanol, or the like, bound by non-covalent intermolecular forces.

[0108] In certain embodiments, the present invention provides a method of treatment for a disease (disorder or condition) affected by aberrant activity of mTOR. The terms "ameliorate" and "treat" are used interchangeably and include both therapeutic and prophylactic treatment. Treating or treatment can mean complete treatment so the subject does not possess any symptoms of the disease or can also mean reducing the symptoms of the disease. Both terms mean decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease (e.g., a disease or disorder delineated herein). Methods of treatment comprise administering a compound of the invention to a subject in need thereof (suffering from a condition or disorder caused by aberrant activity of mTOR). Any disease that is responsive to mTOR inactivation can be treated by a method of the invention. Such diseases include, but are not limited to, neoplastic diseases, cancers, and tumor-prone syndromes. [0109] mTOR inhibitors of the invention are particularly effective against tumors that are PTETV-deficient and/or have abnormalities in the PBK/ AKT pathway, which include certain melanomas, renal cell carcinomas, chronic and acute myeloid leukemias, endometrial cancers, myelomas, and tumors of the prostate, breast, lung, bladder, ovary, pancreas, colon, thyroid and brain. Many such tumors are associated with abnormal signaling through growth factor receptors including, but not limited to, epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR), and insulin-like growth factor 1 receptor (IGF-R). AKT phosphorylation and/or S6K1 expression can be used to predict mTOR inhibitor sensitivity. (See, e.g. Noh, W.C., et al, Clin. Cancer Res. 10, 1013-23).

[0110] mTOR inhibitors of the invention are particularly useful for treatment of neoplastic disease when administered with one or more other cancer therapies, including but not limited to surgery, radiation therapy, chemotherapy, endocrine therapy, hyperthermia, and cryotherapy.

[0111] mTOR inhibitors of the invention can be administered with other agents that are inhibitors of signal transduction, including small molecules and biological agents. As used herein, the terms "chemical agent" and "small molecule" are interchangeable and are used to refer exclusively to substances that have a molecular weight up to, and including, 2000 atomic mass units (Daltons). "Biological agents" are molecules which include proteins, polypeptides, and nucleic acids, and have molecular weights greater than 2000 Daltons.

[0112] In an embodiment of the invention, an mTOR inhibitor of the invention is used in combination with another agent that is a signal transduction inhibitor. An example of such an agent is a receptor tyrosine kinase (RTK) antagonist that neutralizes or reduces that signal transduction activity of a receptor such as an epidermal growth factor receptor (EGFR, HER2) or insulin like growth factor receptor (IGF-R). Such agents include antigen-binding proteins that bind to the extracellular domain of the receptor or to a ligand of the receptor and block binding of the ligand. Ligands for EGFR include, for example, EGF, TGF-α, amphiregulin, heparin-binding EGF (HB-EGF) and betacellulin. EGF and TGF-α are thought to be the main endogenous ligands that result in EGFR-mediated stimulation. Examples of EGFR antagonists that bind EGFR include, without limitation, biological agents such as antibodies (and functional equivalents thereof) specific for EGFR (e.g., cetuximab) or HER2 (e.g., trastuzumab), and chemical agents (small molecules), such as synthetic kinase inhibitors that act directly on the cytoplasmic domain of EGFR (e.g., gefϊtinib, erlotinib). An example of an antibody that binds to IGF-R is IMC-Al 2. [0113] Other non-limiting examples of growth factor receptors involved in tumorigenesis are the receptors for vascular endothelial growth factor (VEGFR-I and VEGFR-2), platelet-derived growth factor (PDGFR), nerve growth factor (NGFR), fibroblast growth factor (FGFR). Examples of inhibitors include sorefenib, which blocks the enzyme RAF kinase, a component of the RAF/MEK/ERK signaling pathway that controls cell division and proliferation and blocks the VEGFR-2/PDGFRβ signaling cascade, and bevacizumab, a monoclonal antibody which binds to VEGF and inhibits signaling through VEFGR.

[0114] In an embodiment of the invention, an mTOR inhibitor described herein is used in combination with another chemotherapeutic drug such as an alkylating agent or an anti-metabolite. Examples of alkylating agents include, but are not limited to, cisplatin, carboplatin, cyclophosphamide, melphalan, and dacarbazine. Examples of anti-metabolites include, but not limited to, methotrexate, doxorubicin, daunorubicin, and paclitaxel, gemcitabine.

[0115] Useful chemotherapeutic agents also include mitotic inhibitors, such as taxanes docetaxel and paclitaxil. Topoisomerase inhibitors are another class of antineoplastic agents that can be used in combination with antibodies of the invention. These include inhibitors of topoisomerase I or topoisomerase II. Topoisomerase I inhibitors include irinotecan (CPT-I l), aminocamptothecin, camptothecin, DX-8951f, and topotecan. Topoisomerase II inhibitors include etoposide (VP- 16), and teniposide (VM -26). Other useful classes include hormones (e.g., tamoxifen, leuprolide, flutamide), proteosome inhibitors (e.g., bortezomib), and antibiotics (e.g., bleomycin, mitomycin). The classes and chemotherapeutic agents identified above are illustrative and non-limiting.

[0116] mTOR antagonist therapy is administered before, during, or after commencing therapy with another agent, as well as any combination thereof, i.e., before and during, before and after, during and after, or before, during and after commencing therapy with the other agent. The combination may provide increased, additive, or synergistic effect. Increased efficiency of the combination often allows for the use of a lower dosage of either or both of the agents than when used alone.

[0117] Treatable tumors include primary and secondary, or metastatic, tumors. The compounds can also be used to treat refractory tumors. Refractory tumors include tumors that fail or are resistant to treatment with chemotherapeutic agents alone, radiation alone or combinations thereof. The mTOR inhibitory compounds are also useful to inhibit growth of recurring tumors, e.g., tumors that appear to be inhibited by treatment with chemotherapeutic agents and/or radiation but recur up to five years, sometimes up to ten years or longer after treatment is discontinued.

[0118] An example of a tumor-prone syndrome is tuberous sclerosis complex (TSC), which was discussed previously in the background of the invention. Another is Peutz-Jeghers syndrome, which is characterized by the presence of multiple gastrointestinal hamartomas. Yet another is lymphangioleiomyomatosis (LAM), a disease of the lung and lymphatics, characterized by pulmonary cysts, recurrent pneumothorax, lymphadenopathy, cystic lymphatic masses, or other manifestations. Another is familial hypertrophic cardiomyopathy.

[0119] Proliferative conditions also include organ hypertrophy, such as familial cardiac hypertrophy, and smooth muscle thickening after vascular injury, such as occurs after placement of a vascular stent, and which can lead to vascular plaque occlusion and atherosclerosis.

[0120] Inhibitors of mTOR are effective as immunosuppressors. Rapamycin has been approved for use as an immunosuppressant in kidney, liver and heart transplantation. Accordingly, the mTOR inhibitors described herein are used to treat autoimmune diseases, such as autoimmune lymphoproliferative disease.

[0121] In an embodiment of the invention, the mTOR inhibitors described herein are used to delay or inhibit an acquired resistance to another drug therapy. Accordingly, an mTOR inhibitor of the invention is coadministered with a drug when resistance to that drug develops, or coadministered throughout treatment with the drug in order to delay or prevent progression to drug resistance.

[0122] For example, acquired resistance is a major problem limiting the benefits of endocrine or hormone-dependent therapy of breast and prostate cancer. Most prostate cancer is initially androgen dependent (AD). Prostate cancer cells initially require androgen for continued proliferation. Response to ablation of testosterone through androgen deprivation therapy (ADT), either surgically (orchiectomy) or medically (GnRH agonists or estrogens), leads to rapid induction of apoptosis of sensitive prostate cancer cells. However, the positive response is followed by a period of growth arrest in which remaining cells tend not to die. After 18-36 months following hormone ablation, growth recurs in 90% of cases. Invariably, surviving cancer cells become androgen independent or unresponsive, and androgen- independent (AI) tumor growth follows. Similarly, a significant proportion of breast malignancies carry receptors responsive to estrogen. In the presence of the hormone, these cancer cells will divide and grow. For this reason, anti-estrogen drugs such as tamoxifen, which blocks estrogen binding, and aromatase inhibitors, which block conversion of testosterone to estrogen, have come to the forefront in the fight against hormone-dependent breast cancer. However, drug resistance often develops after continued exposure to these drugs, and malignant cells proliferate even in the absence of estrogen receptor stimulation.

[0123] The mTOR inhibitors of the invention are inhibitors of signal transduction pathways that are implicated in resistance to endocrine or hormone-dependent therapy. Thus the invention provides a method of delaying or inhibiting resistance to hormone-dependent therapy of prostate and breast cancer, as well as other cancers in signal transduction through pathways that involve mTOR is implicated. In one embodiment, an mTOR inhibitor of the invention is administered in combination with an anti-cancer drug (e.g., a gonadotropin- releasing hormone antagonist for prostate cancer; an anti-estrogen for breast cancer) once resistance to that drug has arisen. In another embodiment, the mTOR inhibitor is coadministered with the anti-cancer drug to delay or prevent progression to drug resistance.

[0124] When administered to a subject, compounds of the present invention can be administered as a pharmaceutical composition containing, for example, a compound and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition.

[0125] One skilled in the art would know that a pharmaceutical composition containing a compound of the present invention can be administered to a subject by various routes including, for example, oral administration; intramuscular administration; intravenous administration; anal administration; vaginal administration; parenteral administration; nasal administration; intraperitoneal administration; subcutaneous administration and topical administration. The composition can be administered by injection or by incubation. The pharmaceutical composition also can be a compound of the invention linked to a liposome or other polymer matrix. Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0126] The present invention also provides kits which comprise a pharmaceutical composition of the invention, wherein said pharmaceutical composition is in a container, and optionally, instructions describing a method of using the pharmaceutical composition for treatment. The included compositions may be lyophilized and packaged with a diluent.

[0127] In certain embodiment, the kits of this invention may comprise separate dosage forms of a compound of this invention and one or more of any of the above-described second therapeutic agents, wherein the compound and second therapeutic agent are associated with one another. The term "associated with one another" as used herein means that the separate dosage forms are packaged together or otherwise attached to one another such that it is readily apparent that the separate dosage forms are intended to be sold and administered together (within less than 24 hours of one another, consecutively or simultaneously).

[0128] The kits of this invention may also comprise a device to administer or to measure out a unit dose of the pharmaceutical composition. Such device may include a syringe and needle if said composition is an injectable composition; a syringe, spoon, pump, or a vessel with or without volume markings if said composition is an oral liquid composition; or any other measuring or delivery device appropriate to the dosage formulation of the composition present in the kit.

[0129] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. It should be understood that the foregoing discussion and examples merely present a detailed description of certain preferred embodiments. It will be apparent to those of ordinary skill in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention. All the patents, journal articles and other documents discussed or cited above are herein incorporated by reference.

EXAMPLES

[0130] Example 1 : In Vitro and Cellular Testing of mTOR Inhibitors

[0131] Several levels of in vitro and in vivo testing are conducted to evaluate the effectiveness of compounds as selective mTOR inhibitors and as potential agents for treatment of TSC using in vitro mTOR and PBK kinase assays. Details of the in vitro assays are as follows. For each compound, dose response curves (0.01 μM to 10 μM) against mTOR, PBKα and PBKβ are constructed. In vitro mTOR assays are performed as previously described (Chiang, G. G. et al, Methods MoI Biol 281, 125-141 (2004)) with modifications. Briefly, the mTORCl complex is immunoprecipitated from FreeStyle 293 (Invitrogen, Carlsbad, CA) cell lysates using a Raptor antibody (Bethyl Laboratories, Montgomery, TX). Immunocomplexes are incubated with a compound of the present invention, added as a solution in dimethylsulfoxide (DMSO) for 30 min. prior to initiating the kinase reaction by adding γ[³²P]ATP and glutathione-S-transferase (GST)-4E-BP1 as substrates. A control reaction in which the inhibitor solution is substituted with an equal volume of DMSO (hereafter called vehicle) is conducted in each series of assays to correct for any effect of solvent. After the reaction is stopped by boiling in SDS sample buffer, the samples are subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). GST-4E- BPl on the gel is visualized by Coomassie blue staining and autoradiography. Radioactive bands are then cut out of the gel and quantified by scintillation counting. These results are used to calculate IC50 values against mTOR for each compound.

[0132] In vitro PBK assays are performed as described by (Ballou, L. M. et al., Journal of Biological Chemistry 278, 23472-23479 (2003)). In this procedure, the pi 10α/p85α complex (PBKα) is purified from baculovirus-infected Sf9 cells as described in (Ballou, L. M. et al., Journal of Biological Chemistry 278, 23472-23479 (2003)). To make the pi 10β/p85α complex (PBKβ), Sf9 cells are coinfected with baculoviruses expressing the human pi lOβ catalytic subunit and p85α regulatory subunits and the PBKβ complex is purified as described for PBKα. Purified PBK is incubated with vehicle or compound for 10 min. prior to initiating the kinase reaction by adding γ[³²P] ATP and phosphatidylinositol as substrates. After the reaction is stopped by adding acidified methanol/chloroform, the samples are subjected to thin layer chromatography (TLC). Radioactive spots corresponding to phosphatidylinositol 3 -phosphate are visualized by autoradiography, cut out of the TLC plate and quantified by scintillation counting. These results are used to calculate IC50 values against PBKα and PBKβ for each compound.

[0133] Compounds that exhibit favorable activity against mTOR and selectivity for mTOR vs. PBKα and PBKβ are further tested in vitro against the two other PBKs (δ and γ). These purified PBK proteins may be purchased from Upstate Biotechnology (Charlottesville, VA). The assays are performed as described above. The compounds are also tested against other PIKK family members (DNA-PK, ATM and ATR) using in vitro kinase assays as described in Chiang, G. G. et al, Methods MoI Biol 281, 125-141 (2004) with modifications. Purified DNA-PK may be purchased from Promega (Madison, WI). ATM and ATR may be obtained by immunoprecipitation from K562 cell lysates using Ab-3 antibody (Calbiochem, San Diego, CA) and ab2905 antibody (Novus Biological, Littleton, CO), respectively. For inhibition assays, the kinases are incubated with increasing concentrations of compound of the present invention and reactions are initiated by adding γ[³²P]ATP and a GST-p53 (1-70 a.a.) fusion protein as substrates. After the reaction is stopped by boiling in SDS sample buffer, the samples are subjected to SDS-PAGE. GST-p53 is visualized by Coomassie blue staining and autoradiography. The radioactive bands are then cut out of the gel and quantified by scintillation counting. IC₅₀ values for each compound against each of these PI3K and PIKK enzymes are calculated.

[0134] Next, in vivo testing is performed on compounds that show good activity and selectivity in vitro. These studies determine whether the compounds can cross the cell membrane and inhibit mTOR signaling. Ratl fibroblasts are serum starved overnight and then incubated with vehicle or increasing concentrations of a compound of the present invention for 20 min. The cells are then stimulated for 10 min. without or with 50 ng/ml PDGF. Phosphorylation of sites modified by mTORCl (S6K T389) or mT0RC2 (Akt S473) is analyzed by Western blotting with phospho-specific antibodies. The blots are stripped and reprobed with general S6K and Akt antibodies to control for equal protein loading. The Western blot bands are quantified using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE) (Lu, Z. et al., J Biol. Chem. 250, 40347-40354 (2005)). IC₅₀ values are calculated for each phosphorylation site. An effective mTOR kinase inhibitor should act like compound 1 of Figure 4 in suppressing S6K T389 and Akt S473 phosphorylation in vivo at low concentrations that are consistent with inhibition of mTOR in vitro (see Figures 7 A and 8A).

[0135] Compounds of the present invention that pass the tests above are evaluated in TSC I^"7" and TSC2^~/~ mouse embryonic fibroblasts (MEFs) obtained from Dr. D. Kwiatkowski (Harvard University, MA). The growth characteristics of these cells and their sensitivity to rapamycin have already been studied (Kwiatkowski, D. J. et al., Human Molecular Genetics 11, 525-534 (2002); (Zhang, H. et al., Journal of Clinical Investigation 112, 1223-1233 (2003)). In addition, it has been shown that long-term rapamycin treatment of these cells upregulates Akt signaling, which is usually abnormally low due to feedback inhibition by mTOR/S6K (Yang, Q. et al., Proceedings of the National Academy of Sciences USA 103, 6811-6816 (2006); Shah, O. J. et al., Current Biology 14, 1650-1656 (2004)). This response to rapamycin would be undesirable in a clinical setting because feedback inhibition of Akt is thought to limit the growth and contribute to the benign nature of TSC-related tumors (Manning, B. D. et al., Genes and Development, 19, 1773-1778 (2005)). To determine if mTOR kinase inhibitors and rapamycin have different effects on Akt, cells are cultured in complete growth medium for 4 days with a compound of the present invention (dose based on its IC50 against mTOR determined above), 50 nM rapamycin or vehicle. Cell lysates are analyzed by Western blotting to detect S6K phospho-T389, Akt phospho-S473 and total levels of both proteins. Since mTOR inhibitors block both mTORCl and mT0RC2, it is expected that phosphorylation of both S6K and Akt will be reduced. In contrast, long term treatment with rapamycin should reduce S6K phosphorylation but increase Akt phosphorylation in these TSC knockout cells as previously reported (Yang, Q. et al., Proceedings of the National Academy of Sciences USA 103, 6811-6816 (2006); Shah, O. J. et al., Molecular and Cellular Biology 26, 6425-6434 (2006)).

[0136] Next, the effect of an mTOR inhibitor of the present invention on three clinically relevant growth-related responses of TSCl^"7" and TSC2 ^{~ ~} MEFs is examined following treatment with increasing concentrations of compound, 50 nM rapamycin, or vehicle for 4 days.

[0137] The effect of the compound on cell proliferation is examined using a colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) proliferation assay. Cells are plated in triplicate at 3,000 cells/well in 96-well plates and the next day they are treated with a compound of the present invention or vehicle. After 4 days the number of viable cells is determined using the MTT assay as previously described (Mosmann, T., Journal of Immunological Methods. 65, 55-63 (1983)). The results are read on a plate reader and the effect of the compound of the present invention expressed as the percentage of viable cells relative to vehicle-treated controls. The results are used to calculate the IC₅₀ for each compound of the present invention. It is expected that rapamycin will have a small inhibitory effect on proliferation at this concentration (Zhang, H. et al., Journal of Clinical Investigation. 112, 1223-1233 (2003)). A larger effect of the mTOR inhibitor of the present invention might be expected because of its dual inhibition of mTORCl and mTORC2. [0138] Whether a compound of the present invention induces cell death is then tested. Cleavage of genomic DNA during apoptosis yields single strand breaks whose free 3'-OH ends can be labeled with fluorescein-tagged dUTP using terminal deoxynucleotidyl transferase (TdT). The level of apoptosis is measured by TdT -mediated dUTP nick end labeling (TUNEL) followed by flow cytometry as previously described in (Ballou, L. M. et al, Journal of Biological Chemistry 275, 4803-4809 (2000)). Because Akt activates survival pathways, it is expected that rapamycin-treated cells will exhibit a relatively low level of apoptosis. The mTOR inhibitors of the present invention may induce a higher rate of cell death because of their suppression of mT0RC2 and Akt. A strong cytotoxic effect of the compound on TSC cells would be clinically more desirable than a cytostatic effect detected in the experiment above.

[0139] The effect of the compound on cell size is then analyzed. Inappropriate upregulation of mTOR signaling due to the loss of TSCl or TSC2 can lead to an increase in cell size. Subependymal giant cell astrocytomas and cortical tubers containing abnormally enlarged neurons and glial cells can develop in the brains of TSC patients. Although these lesions are not malignant, they can cause seizures, hydrocephalus and mental retardation. Rapamycin treatment has been shown to cause regression of astrocytomas in TSC patients, and this effect may be due in part to a reduction in cell size. (Franz, D. N. et al., Annals of Neurology 59, 490-498 (2006)). It is expected that both the mTOR inhibitors of the present invention and rapamycin will reduce the size of TSC I^"7" and TSC^"7" MEFs. This result would suggest the possible utility of an mTOR inhibitor of the present invention in treating central nervous system manifestations of TSC, even if the compound does not exhibit dramatic cytostatic or cytotoxic effects. Following drug treatment as described above, MEFs are trypsinized and suspended in a solution of growth medium plus Isoton II Diluent (Beckman). Average cell diameters and volumes are determined using a Multisizer 3 Coulter Counter (Beckman). It is expected that variability in cell size will be relatively large; therefore at least 10,000 cells are measured for each experimental condition to minimize sampling error.

[0140] Finally, the most promising compounds of the invention are tested against a large panel of protein kinases (KINASEPROFILER™ Selectivity Screening Service, Upstate, Charlottesville, VA) to exclude those that have a broad inhibitory spectrum.

[0141] In vivo testing of the most promising mTOR inhibitor(s) of the present invention (based on the studies above) is conducted by testing the compounds in TSCl^"7" mice (available from NCI). These mice develop many features of the human disease and thus are good animal models for the treatment of TSC.

[0142] Example 2: Testing Of Certain Compounds Of The Present Invention

[0143] Compound 1 of Figure 4 and several compounds of the present invention, including those of structure I wherein B is Bl and A includes moieties shown in Figure 6 were tested for inhibition of the kinase activity of mTOR in vitro. Results for compound 1 illustrate preferred properties for a selective mTOR kinase inhibitor. First, compound 1 inhibits mTOR activity in a dose-dependent manner, which was confirmed using an in vitro kinase assay with γ[³²P]ATP and GST-4E-BP1 as substrates (Figure 7A). GST is glutathione-S-transferase and 4E-BP1 is eukaryotic initiation factor 4E binding protein. Compound 1 is a poor inhibitor of PBKs as compared with LY294002 (Figure 7B). The PBKs were assayed using γ[³²P]ATP and phosphatidylinositol as substrates. (Ballou, L. M., et al, Biochem. J. 394, 557-562 (2006)).

[0144] Next, the effect of compound 1 of Figure 4 on mTOR signaling in cultured cells was tested. To assess intracellular mTOR activity, the phosphorylation of S6K (ribosomal protein S6 kinase) and Akt was analyzed on Western blots probed with phospho- specifϊc antibodies that recognize residues phosphorylated by mTOR (S6K T389 and Akt S473). Ratl fibroblasts were pretreated with inhibitors and then stimulated with platelet- derived growth factor (PDGF). Compound 1 of Figure 4 and LY294002 strongly blocked both S6K T389 and Akt S473 phosphorylation due to their inhibitory effect on mTOR (Figure 8A). As expected, rapamycin only inhibited the mTORCl site (S6K T389), and compound 1 of Figure 4 had little effect on Akt T308, which is phosphorylated by PDKl (phosphoinositide-dependent kinase) following activation of PBK (Figure 8A).

[0145] The change in Akt phosphorylation levels when MCF7 breast cancer cells are treated for an extended time with compound 1 (of Figure 4) was also examined. As has been reported by others (O'Reilly, K. E. et al., Cancer Res 66, 1500-1508 (2006)), treatment of these cells for 24 hours with rapamycin led to increased phosphorylation of Akt S473 (Figure 8B). Inhibition of mTORCl by rapamycin is thought to turn off a negative feedback loop that normally suppresses Akt activity. Since Akt provides a survival signal, rapamycin treatment may result in enhanced tumor growth and survival. In contrast, compound 1 of Figure 4 did not induce Akt phosphorylation because it inhibits both mTORCl and mT0RC2 (Figure 8B). As expected, rapamycin and compound 1 of Figure 4 markedly reduced S6K T389 phosphorylation (Fig. 8B). Compound 1 of Figure 4 also blocked MCF7 breast cancer cell proliferation by 50% at a concentration between 10 and 25 μM as measured by MTT assays (Figure 8C). These results indicate that compound 1 of Figure 4 exhibits the expected properties of an mTOR kinase inhibitor both in vitro and in vivo. Thus compound 1 of Figure 4 and LY294002 were used as lead structures for the design of potent and selective inhibitors of mTOR, which are compounds of the present invention.

[0146] Other compounds of the present invention (Structure I, wherein B is Bl and A is selected from Al-AlO) have also been subjected to preliminary screening as inhibitors of mTOR and of PBK, with data shown below each carbon-8 substituent in Figure 6. The compound having structure I wherein A is Al and B is Bl, which combines the structure of LY294002 with the heteroatom arrangement of compound 1 of Figure 4 was a modest inhibitor of mTOR but exhibited no measurable inhibition of PBKα at 5 μM. The compound having structure I wherein A is A2 showed significantly enhanced inhibition of mTOR relative to the compound having structure I wherein A is Al but also exhibited no inhibition of PBK. Interestingly, the analog having the original chromenone nucleus of LY294002 but also having the carbon-8 phenyl substituent replaced with the o-naphthyl substituent as in the compound having structure I wherein A is A2 was reported to have more than 10-fold decreased inhibition of DNA-PK relative to LY294002 (Hardcastle, I. R. et al, J Med Chem 48, 7829-7846 (2205)). The results point to the compound having structure I wherein A is A2 as a good candidate for more selective mTOR inhibition. The compound having structure I wherein A is A3 exhibited similar activity to the compound having structure I wherein A is Al against mTOR while the compound having structure I wherein A is A4 was much less active. The compound having structure I wherein A is A5 exhibited the most potent inhibition of mTOR among this group, but also strongly inhibited PBK. Thus, of the top row of Figure 6, the compound having structure I wherein A is A2 is a compound of interest. Among the second row, the compound having structure I wherein A is A6 exhibited potent inhibition of mTOR, similar to the compound having structure I wherein A is A2. It is noteworthy that while these compounds (having structure I wherein A is A2 or A6) exhibit similarly potent inhibition of mTOR, they are also structurally very similar, with compound (A6) simply lacking the 2-carbon bridge to form the second ring of compound (A2). Again, the analog having the same substituent as the compound having structure I wherein A is A7 at carbon-8 of the chromenone nucleus of LY294002 was reported to have 6.5-fold lower activity in inhibition of DNA-PK relative to LY294002 having the carbon-8 phenyl group. (Hardcastle, I. R. et al, J Med Chem 48, 7829-7846 (2205)). The compounds having structure I wherein A is A6, A7, A8 or A9 were prepared as isosteres of the compound having structure I wherein A is A6 having a ring nitrogen to potentially interact with the protonated amine group of K2171 in mTOR. All three had improved solubility relative to compound (A6). The compound having structure I wherein A is A7 showed the best activity of these three, and indeed this analog is predicted by computer modeling to have the ring nitrogen closest to the K2171 amine group. However, these three analogs also exhibited strong inhibition of PBK. Computer modeling suggests that the pyridine nitrogen atom of these analogs may form a hydrogen bond with the side chain hydroxyl group of T856 of PBKα (corresponds to T887 of PBKγ). The amide isostere (the compound having structure I wherein A is AlO) of the olefin A6 (the compound having structure I wherein A is A6) exhibited potent inhibition. The isomeric amide having the orientation of the nitrogen and carbonyl reversed was also prepared but showed minimal inhibition of mTOR. The results of these assays are shown in Table 1.

[0147] The analogs designed to target the C2243 thiol group have also been investigated. The chloride (the compound having structure II wherein B is B2) did not exhibit any detectable inhibition of mTOR at 25 μM. However, the thiol (the compound having structure II wherein B is B9) inhibited mTOR with an IC50 value of 0.3 μM, clearly the most potent inhibition of any analog tested or reported in the literature. As such, the compound having structure II wherein B is B9 is a preferred compound of the invention. These results for Structure II are depicted in Table 2:

Claims

We claim:

wherein:

A is selected from H, alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, cycloalkenyl, halo, aryl, -(C_1-6 alkyl)-aryl, -(C₂-₆ alkenyl)-aryl, -NH-C(O)-aryl, and -C(O)-NH-aryl;

R¹ is selected from H, alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, cycloalkenyl, halo, aryl, -(Ci_6 alkyl)-aryl, and -(C 2-6 alkenyl)-aryl;

-(CH₂VO-R⁴¹, -(CH₂VN(R⁴²XR⁴³), -(CH₂)_Ω-N(R⁴¹)-(CH₂),C(O)R⁴⁴, -(CH₂)_Ω-SR⁴¹, -(CH₂)_Ω-C(O)R⁴⁴, -(CH₂)_Ω-C(O)-(CH₂)_δOR⁴¹, -(CH₂)_Ω-C(O)-(CH₂)_δN(R⁴²)(R⁴³), -(CH₂)_ΩO-C(O)R⁴⁴, -(CH₂)_ΩS-C(O)R⁴⁴, -(CH₂)_ΩOC(O)-(CH₂)_δN(R⁴²)(R⁴³), -(CH₂VAs(R⁴⁴XR⁴⁴), -(CH₂VCH=CH-(CH₂VR⁴⁰, and -(CH₂)_Ω-C(=CH₂)-(CH₂)_δ-R⁴⁰;

R⁴¹ is selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl and a heterocyclic group; each R⁴² and R⁴³ are independently selected from H, alkyl, cycloalkyl, cycloalkenyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic group; or R and R may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom and may be optionally substituted with up to three substituents selected from halo, CN, NO₂, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic group; each R⁴⁴ is independently selected from H, alkyl, -OH, -O-alkyl, -O-aryl, -O-aralkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl and a heterocyclic group; a is O to 4; b is 0 to 4; and n is 0, 1 or 2.

2. The compound of claim 1, wherein R² is selected from H, halo, amino, hydroxyl, methyl and trifluoromethyl.

3. The compound of claim 2, wherein R² is H.

4. The compound of claim 1 , wherein R .3 i •s selected from H, halo, amino, hydroxyl, methyl and trifluoromethyl.

5. The compound of claim 4, wherein R³ is H.

6. The compound of claim 1, wherein A is selected from aryl, -(C_1-6 alkyl)-aryl, -(C₂-6 alkenyl)-aryl, and -NH-C(O)-aryl.

7. The compound of claim 1, wherein R¹ is selected from H, halo, and alkyl.

8. The compound of claim 7, wherein R¹ is H.

9. The compound of claim 1, wherein R⁴ is selected from halo, amino, hydroxyl,

- 42_wr> 43_\ alkyl, alkenyl, alkynyl, cycloalkyl, -(CH₂)_Ω-O-R , -(CH₂)_Ω-N(R^4Z)(R^4J), -(CH₂)_Ω-N(R⁴¹)-(CH₂),C(O)R⁴⁴, -(CH₂)_Ω-SR⁴¹, -(CH₂)_Ω-C(O)R⁴⁴, -(CH₂)_Ω-C(O)-(CH₂),OR⁴¹,

> 42_λ > 43 44 > 44

-(CH₂)_Ω-C(O)-(CH₂),N(R^4Z)(R ), -(CH₂)_ΩO-C(O)R⁴⁴, -(CH₂)_ΩS-C(O)R⁴

.42_wr> 43 40

-(CH₂)_ΩOC(O)-(CH₂)_δN(R^4Z)(R ), -(CH₂)_Ω-CH=CH-(CH₂)_δ-R^4υ, and

-(CH₂)_Ω-C(=CH₂)-(CH₂),-R 4⁴0^U.

10. The compound of claim 1, having the formula:

wherein A is selected from:

Al A2 A3

A6

A4 A5

All A12 A13 A14

and B is selected from;

Bl B2 15

B6 B7

B9

B13

B12

and wherein X is selected from CN, NO₂, and CO₂CH₃.

11. The compound of claim 10, wherein A is A2 or A6.

12. The compound of claim 10, wherein A is Al and B is Bl.

13. The compound of claim 10, wherein A is A2 and B is B 1.

14. The compound of claim 10, wherein A is A3 and B is Bl.

15. The compound of claim 10, wherein A is A4 and B is B 1.

16. The compound of claim 10, wherein A is A5 and B is B 1.

17. The compound of claim 10, wherein A is A6 and B is B 1.

18. The compound of claim 10, wherein A is A7 and B is B 1.

19. The compound of claim 10, wherein A is A8 and B is B 1.

20. The compound of claim 10, wherein A is A9 and B is B 1.

21. The compound of claim 10, wherein A is Al 0 and B is B 1.

22. A compound of the formula:

wherein:

-(Ci_6 alkyl)-aryl, and -(C 2-6 alkenyl)-aryl;

R is selected from H, halo, amino, hydroxyl, lower alkyl, lower alkenyl, and lower alkynyl;

-(CH₂VO-R⁴¹, -(CH₂VN(R⁴²XR⁴³), -(CH₂)_Ω-N(R⁴¹)-(CH₂)_δC(O)R⁴⁴, -(CH₂)_Ω-SR⁴¹, -(CH₂)_Ω-C(O)R⁴⁴, -(CH₂)_Ω-C(O)-(CH₂),OR⁴¹, -(CH₂)_Ω-C(O)-(CH₂)_δN(R⁴²)(R⁴³), -(CH₂)_ΩO-C(O)R⁴⁴, -(CH₂)_ΩS-C(O)R⁴⁴, -(CH₂)_ΩOC(O)-(CH₂)_δN(R⁴²)(R⁴³), -(CH₂VAs(R⁴⁴XR⁴⁴), -(CH₂VCH=CH-(CH₂VR⁴⁰, and -(CH₂)_Ω-C(=CH₂)-(CH₂)_δ-R⁴⁰;

R⁵ is selected H, halo, -OH, amino, alkyl, -O-alkyl, -O-aryl, -O-aralkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, -(C_1-6 alkyl)-aryl, and -(C 2-6 alkenyl)-aryl, and a heterocyclic group; a is 0 to 4; b is 0 to 4; m is 1 or 2; and p is 0 to 3.

23. The compound of claim 22, wherein R is selected from H, halo, amino, hydroxyl, methyl and trifluoromethyl.

24. The compound of claim 23, wherein R is H.

25. The compound of claim 22, wherein R³ is selected from H, halo, amino, hydroxyl, methyl and trifluoromethyl.

26. The compound of claim 25, wherein R³ is H.

27. The compound of claim 22, wherein R⁴ is selected from halo, amino, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, -(CH₂)_Ω-O-R⁴¹, -(CH₂)_Ω-N(R⁴²)(R⁴³), -(CH₂)_Ω-N(R⁴¹)-(CH₂),C(O)R⁴⁴, -(CH₂)_Ω-SR⁴¹, -(CH₂)_Ω-C(O)R⁴⁴, -(CH₂)_Ω-C(O)-(CH₂)_δOR⁴¹, -(CH₂)_Ω-C(O)-(CH₂)_δN(R⁴²)(R⁴³), -(CH₂)_ΩO-C(O)R⁴⁴, -(CH₂)_ΩS-C(O)R⁴⁴, -(CH₂)_ΩOC(O)-(CH₂),N(R⁴²)(R⁴³), -(CH₂)_Ω-CH=CH-(CH₂),-R⁴⁰, and -(CH₂)_Ω-C(=CH₂)-(CH₂)_δ-R⁴⁰.

28. The compound of claim 22, having the formula:

wherein B is selected from:

B6 B7

B13

B12

and wherein X is selected from CN, NO₂, and CO₂CH₃.

29. The compound of claim 22, wherein B is B2.

30. The compound of claim 22, wherein B is B3.

31. The compound of claim 22, wherein B is B4.

32. The compound of claim 22, wherein B is B5.

33. The compound of claim 22, wherein B is B6.

34. The compound of claim 22, wherein B is B7.

35. The compound of claim 22, wherein B is B8.

36. The compound of claim 22, wherein B is B9.

37. The compound of claim 22, wherein B is B 10.

38. The compound of claim 22, wherein B is B 11.

39. The compound of claim 22, wherein B is B 12.

40. The compound of claim 22, wherein B is B 13.

41. The compound of claim 22, wherein B is B14.

42. The compound of claim 22, wherein B is B 15.

43. The compound of claim 22, wherein B is B 16.

44. The compound of claim 22, wherein B is B 17.

45. The compound of claim 22, wherein B is B 18.

46. A compound of any one of claims 1-45, which inhibits mTOR.

47. A compound of claim 46 which inhibits mTOR with an IC50 of less than about 2μM.

48. A compound of claim 47 which inhibits PBK with an IC50 of more than about 25 μM.

49. A pharmaceutical composition comprising a compound of any of claims 1-45 and a pharmaceutically acceptable carrier.

50. A method of inhibiting mTOR activity in a cell which comprises providing to said cell a compound of any one of claims 1-45.

51. A method of inhibiting mTOR activity in a subject which comprises providing to the subject an effective amount of a compound of any one of claims 1-45.

52. A method of treating a proliferative condition in a subject comprising administering to the subject an effective amount of a compound of any one of claims 1-45.

53. The method of claim 52, wherein the proliferative condition is a tumor-prone syndrome.

54. The method of claim 53, wherein the tumor-prone syndrome is tuberous sclerosis complex.

55. A method of treating a neoplastic disease in a subject comprising administering to the subject an effective amount of a compound of any one of claims 1-45.

56. The method of claim 55, wherein the neoplastic disease is cancer.

57. The method of claim 55, wherein the proliferative condition a neuronal tumor, breast cancer, prostate cancer, acute myelogenous leukemia, lung cancer, pancreatic cancer, colon cancer, renal cancer, or myeloma.

58. A method of delaying or inhibiting an acquired resistance to a drug used for treating a proliferative condition or neoplastic disease in a subject, comprising coadministering with the drug an effective amount of a compound of any one of claims 1-45.

59. The method of any one of claims 51 to 58, wherein the subject is a mammal.

60. The method of any one of claims 51 to 58, wherein the subject is a human.

61. Use of a compound of any one of claims 1 -45 in the manufacture of a medicament for inhibiting mTOR in a subject.

62. Use of a compound of any one of claims 1 -45 in the manufacture of a medicament for treating a proliferative condition in a subject.

63. The use according to claim 62, wherein the proliferative condition is a tumor- prone syndrome.

64. The use according to claim 63, wherein the tumor-prone syndrome is tuberous sclerosis complex.

65. Use of a compound of any one of claims 1 -45 in the manufacture of a medicament for treating a neoplastic disease in a subject.

66. The use according to claim 65, wherein the proliferative condition is cancer.

67. The use according to claim 65, wherein the proliferative condition a neuronal tumor, breast cancer, prostate cancer, acute myelogenous leukemia, lung cancer, pancreatic cancer, colon cancer, renal cancer, or myeloma.

68. Use of a compound of any one of claims 1 -45 in the manufacture of a medicament for delaying or reversing an acquired resistance to a drug used for treating a proliferative condition or neoplastic disease in a subject.

69. A kit for treatment of a proliferative condition or neoplastic disease in a subject comprising, said kit comprising a compound of any one of claims 1-45, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, and a container for containing said compound.

70. The kit of claim 69, wherein said compound is lyophilized and at least one further component part comprises a diluent.

71. The kit of claim 69, which further comprises an inhibitor of a receptor tyrosine kinase.

72. The kit of claim 71 , wherein the receptor tyrosine kinase is epidermal growth factor receptor.

73. The kit of claim 71 , wherein the receptor tyrosine kinase is insulin-like growth factor 1 receptor.