SMALL MOLECULE RAS LIGANDS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Patent Application Serial No. 62/155,352, filed on April 30, 2015 which application is incorporated by reference herein in its entirety.

GOVERNMENT FUNDING

[0002] This invention was made with government support under grant no. 5R01 CA161061 -04 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF INVENTION

[0003] The present invention provides, inter alia, small molecule RAS inhibitors and compositions containing such compounds. Methods for using such compounds or compositions are also provided.

BACKGROUND OF THE INVENTION

[0004] A relatively unexplored subset of therapeutic targets encompass proteins capable of eliciting biological events through protein-protein interactions (PPIs) (Hopkins et al.). While some PPIs have proven tractable to impediment by small molecules (e.g. p53-Mdm2) (Vassilev), the majority of PPIs have yet to succumb to small molecule modulation, especially in the context of high-throughput screening of standard chemical libraries (Nero). Typical of this class are the RAS GTPases, a coveted target in cancer biology due to their high prevalence and essentiality in some lethal malignancies. RAS mutations are the most frequent gain of function oncogenic mutations in human cancer, with the KRAS isoform exhibiting the strongest correlation (Prior et al.). Present in 20-30% of all malignancies, it is found at even higher rates in three of the top four most lethal cancers: pancreatic (90%), colon (45%), and lung (35%) (Downward). RAS proteins have proven historically resistant to small molecule inhibition, as active site nucleotide affinity (pM range) precludes competitive inhibition strategies (John et al.). Coupled with a protein topography that fails to present binding regions for effective allosteric modulation, clinical and pre-clinical evaluation of what is considered to be the "holy grail" of cancer targets— RAS inhibitors— has been limited.

[0005] RAS proteins play a critical role in several signal transduction pathways regulating cell growth and differentiation. Functioning as a binary switch, RAS GTPases transition from an inactive GDP-bound conformation to an active GTP- bound form mediated by guanine nucleotide exchange factors (GEFs, namely sons of sevenless, SOS) and GTPase activating proteins (GAPs) (Hall et al.). GTP binding enables a conformational change that permits RAS interaction with its effector proteins. Mutations resulting in the impairment of RAS inactivation contribute towards reinforcing downstream signaling pathways that result in a malignant phenotype. Oncogenic signal transduction is mainly supported by RAS effectors RAF, RALGDS, and PI3K (Downward, Pacold et al., Huang et al., Block et al.) (FIG. 1 ). Targeting these particular PPIs presents an intriguing approach towards mitigating RAS signaling in the context of developing cancer therapeutics.

[0006] It is important to note that not all endeavors towards small molecule inhibition of RAS signaling have been futile. Over the past two years, several groups have generated substrates exhibiting moderate, yet appreciable, RAS inhibition (See, e.g. FIG. 2). Several reports of small molecule RAS-SOS inhibitors were published in 2012, typically employing fragment-based drug discovery approaches in order to prevent activation of RAS to its GTP-bound form. Maurer et al. and Sun et al. independently pursued NMR-based fragment screens as an initial method towards identifying molecules capable of binding to RAS, such as compounds 1 and 2 (Sun et al., Maurer et al.) (See, e.g. FIG. 2A). The hits typically incorporated indole-based scaffolds, binding in the conserved site of RAS-SOS interaction near L56. The most potent lead compound described by Maurer et al. (Kd = 1 .5 mM) was the only substrate that exhibited activity in vitro, attenuating EGF-stimulated HEK- 293T cell proliferation. Efforts to elaborate these fragments to a more potent inhibitor were unsuccessful. A cell-permeable, synthetic a-helix (compound 3, FIG. 2A), based on the interacting portion of SOS, was developed as a means to interfere with the RAS-SOS interaction (Patgiri et al.). The helix targeted a stretch of residues contained within the switch I region of RAS, incorporating a hydrogen-bond surrogate to facilitate plasma stability and cell permeability. The peptidomimetic bound nucleotide-free RAS with a KD of 28 μΜ and GDP-bound RAS to a lesser extent (158 μΜ), successfully abrogated RAS signaling in EGF-stimulated HeLa cells. The relatively underwhelming potency of these compounds impedes further development towards clinical candidates. Although these approaches were potentially valuable in cancers reliant on SOS-mediated activation of wild-type RAS for proliferation, oncogenic RAS would remain largely unaffected through this mechanism.

[0007] Shima et al. examined an in silico approach towards inhibiting surface interaction between RAS and RAF, generating compounds, such as compound 4 (FIG. 2A), with inhibitory constants (Ki) in the 46 to 733 μΜ range and activity in a colon cancer xenograft model (Shima et al.). Again, the modest potency limits further development, but provides an early example of targeting RAS-specific PPIs. Ostrem et al. more recently developed an approach towards targeting KRAS ^G12C by exploiting the reactivity of the nucleophilic cysteine residue native to the mutant isoform Ostrem et al.). They used a fragment-based screening strategy to design covalent inhibitors with considerable success. Their most potent inhibitor (compound 5, FIG. 2A) was shown to irreversibly prevent the RAS-RAF interaction selectively at 100 μΜ in a KRAS ^G12C mutant cell line when compared to cells expressing wild-type KRAS. A drawback is the specificity for the KRAS ^G12C mutant, which represents a limited number of malignant RAS isoforms. In vitro assays also revealed promiscuous binding to other protein targets, a potential pitfall of incorporating the reactive electrophilic moiety.

[0008] Aside from binding RAS directly, drug discovery programs have targeted post-translational modifications, such as farnesylation and palmitoylation, as modes of inhibiting RAS signaling. RAS requires post-translational modifications in order to facilitate membrane trafficking where it can induce downstream signaling (Ahearn et al.). Farnesyltransferase inhibitors (FTI) have been developed as ligand biomimetics of farnesyl pyrophosphate (FPP) analogs, as well as peptide mimetics of the RAS hydrophobic tail region necessary for membrane trafficking (terminal CAAX motif) (Baker et al.). While two inhibitors of this class, tipifarnib (6) and lonafarnib (7) (See FIG. 2B), progressed to phase II clinical trials, compensatory mechanisms led to disappointing in vivo efficacy, while simultaneous targeting of such mechanisms forfeited any appreciable therapeutic window (Mesa, Ravoet et al.).

[0009] Following the disappointing outcome of the FTIs, targeting enzymes involved in RAS localization post-prenylation was explored. PDE65 was recently revealed to be a farnesyl-binding chaperone that facilitates the trafficking and signaling of RAS (Cox et al.). High Throughput Screening (HTS) was performed with biotinylated and farnesylated KRAS4B against His-tagged PDE65 in an attempt to discover molecules that disrupt this interaction. Several hits containing a benzimidazole scaffold were identified and further optimized, generating inhibitors with activity in a mouse xenograft models (deitarasin, 8, FIG. 2B) (Zimmerman et al.). Although these molecules were not pursued further in a clinical setting, this demonstrated HTS as a viable strategy for treating RAS mutated tumors, while circumventing drawbacks of FTI inhibition.

[0010] Overall, the modest progression of RAS inhibitors into human clinical trials reflects an inability to effectively abrogate the multiple facets of RAS signaling. This is likely due to either suboptimal potency or prevailing compensatory mechanisms. Thus, there exists an unmet need for compounds that selectively bind a RAS protein, particularly an oncogenic mutant of a RAS protein. The present invention details an approach capable of enabling the development of small molecules with sub-micromolar RAS affinity, while efficiently mitigating the multitude of downstream signaling pathways by targeting key protein-protein interactions.

SUMMARY OF THE INVENTION

[0011] One embodiment of the present invention is a compound that selectively binds a RAS protein at three or more sites, which compound selectively binds to a first site on the RAS protein that comprises at least one amino acid near L56.

[0012] Another embodiment of the present invention is a compound having the structure of formula (V): wherein:

R ₄ is selected from a group consisting of no atom, H, aryl, halide, Ci _-4 aliphatic, and -0-Ci _-4 aliphatic, wherein the Ci _-4 aliphatic is optionally substituted with one or more halide;

R ₈ is selected from a group consisting of no atom, H, alkyl, and aryl, wherein the alkyl is optionally substituted with a group consisting of halide, ether, and combinations thereof, and the aryl is optionally substituted with one or more groups consisting of halide, ether, Ci _-4 alkyl, and combinations thereof;

R ₉ is selected from a group consisting of no atom, H, and aryl optionally substituted with a group consisting of ether, halide, and combinations thereof;

Ri2, Ri 3, Ri4, and R ₅ are independently selected from a group consisting of H and -X-E, wherein X is selected from a group consisting of a bond, NH and 0, and wherein E is an electrophile;

m and n are independently selected from integers between 0-5; and ring A is a heterocycle with at least 1 ring nitrogen and optionally substituted with Ci _-4 alkyl or a halide,

or a crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0013] Another embodiment of the present invention is a compound having the structure of formula (VI):

R ₄ is selected from a group consisting of no atom, H, aryl, halide, Ci _-4 aliphatic, and -0-Ci _-4 aliphatic, wherein the Ci _-4 aliphatic is optionally substituted with one or more halide;

R ₈ and R-n are independently selected from a group consisting of no atom, H, alkyi, and aryl, wherein the alkyi is optionally substituted with a group consisting of halide, ether, and combinations thereof, and the aryl is optionally substituted with one or more groups consisting of halide, ether, d. 4alkyl, and combinations thereof; R-io is selected from a group consisting of no atom, H, halide, and Ci _-4 aliphatic;

Ri2, Ri3, Ri4, and Ri ₅ are independently selected from a group consisting of H and -X-E, wherein X is selected from a group consisting of a bond, NH and 0, and wherein E is an electrophile;

m and n are independently selected from integers between 0-5; and ring A is a heterocycle with at least 1 ring nitrogen and optionally substituted with Ci _-4 alkyl or a halide,

or a crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0014] Another embodiment of the present invention is a compound selected from a group consisting of

and crystalline forms, hydrates, or pharmaceutically acceptable salts thereof.

[0015] Another embodiment of the present invention is a compound having the structure:

or a crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0016] Another embodiment of the present invention is a compound having the structure:

or a crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0017] Another embodiment of the present invention is a compound having the structure:

or a crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0018] Another embodiment of the present invention is a compound having the structure:

or a crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0019] Another embodiment of the present invention is a compound having the structure: wherein R ₂ , R13, Ri ₄ , and R ₅ are independently selected from a group consisting of H and -X-E, wherein X is selected from a group consisting of a bond, NH and 0, and wherein E is an electrophile,

or a crystalline form, hydrate, or pharmaceutically acceptable salt thereof,

with the provisio that at least one of R ₂ , R13, Ri ₄ , and R ₅ are not H.

[0020] Another embodiment of the present invention is a compound selected from a group consisting of

and crystalline forms, hydrates, or pharmaceutically acceptable salts thereof.

[0021] Another embodiment of the present invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the present invention.

[0022] Another embodiment of the present invention is a method for treating or ameliorating the effects of a disease associated with altered RAS signaling in a subject comprising administering to the subject an effective amount of a compound of the present invention.

[0023] Another embodiment of the present invention is a method for treating or ameliorating the effects of a disease associated with altered RAS signaling in a subject comprising administering to the subject an effective amount of a pharmaceutical composition of the present invention.

[0024] Another embodiment of the present invention is a method for effecting cancer cell death comprising contacting a cancer cell with an effective amount of a compound of the present invention.

[0025] Another embodiment of the present invention is a kit for treating or ameliorating the effects of a disease related to altered RAS signaling in a subject in need thereof, the kit comprising an effective amount of a compound or pharmaceutical composition of the present invention packaged together with instructions for its use. [0026] Another embodiment of the present invention is a kit for treating or ameliorating the effects of a cancer in a subject in need thereof, the kit comprising an effective amount of a compound or pharmaceutical composition of the present invention, packaged together with instructions for its use.

[0027] Another embodiment of the present invention is a composition comprising a compound of the present invention.

[0028] Another embodiment of the present invention is a compound that selectively binds a RAS protein at three or more sites, wherein when the compound is bound to the RAS protein, the compound binds to L56 of the RAS protein and blocks the binding of SOS to the RAS protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0030] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0031] FIG. 1 is a diagram showing RAS-signaling pathways. RAS downstream signaling through effector proteins PI3K, RAF, and RALGDS promotes cell proliferation.

[0032] FIG. 2 shows chemical structures of previously developed RAS inhibitors targeting (A) effector regions and (B) posttranslational modifications. [0033] FIG. 3 shows the sites of targeted RAS-effector interactions, regions (A) switch I (red) and switch II (blue) regions (B) D38 (green) and A59 (purple).

[0034] FIG. 4 demonstrates virtual screening efforts targeting the additional shallow pocket adjacent to the trifluoromethylphenyl ring near the L56 site in (A). The top-scoring fragment from an in silico screen is shown in (B).

[0035] FIG. 5 shows targeting of the L56 site. (A) depicts the virtual anticipated pose of a fully elaborated 3-site compound docked into 4DSN. (B) shows ligand-receptor interactions at L56.

[0036] FIG. 6 shows the synthesis of the novel 3-site compound 04SBT04 (Scheme 1 ).

[0037] FIG. 7 A and B shows the synthesis of electrophilic fragments to assess covalent inhibiton at L56 site (Scheme 2).

[0038] FIG. 8 shows additional structures involved in the synthesis reactions of Schemes 1 and 2.

DETAILED DESCRIPTION OF THE INVENTION

[0039] One embodiment of the present invention is a compound that selectively binds a RAS protein at three or more sites, which compound selectively binds to a first site on the RAS protein that comprises at least one amino acid near L56. [0040] As used herein, "selectively binds", and grammatical variations thereof, means a binding reaction between two molecules that is at least two times the background and more typically more than 10 to 100 times background molecular associations under physiological conditions. Likewise, compounds "selective" for a given form of a RAS protein may exhibit molecular associations under physiological conditions at least two times the background and more typically more than 10 to 100 times background.

[0041] As used herein, "RAS proteins" include all RAS isoforms, which are members of a family of GTPase proteins frequently mutated in numerous cancers. The terms, "isoform" and grammatical variations thereof, refer to functionally similar proteins that have a similar, but not identical amino acid sequence, and may also be differentially post-translationally modified. RAS isoforms include, but are not limited to HRAS, KRAS, and NRAS. The HRAS, KRAS, and NRAS proteins are highly homologous to one another and have similar mechanisms of action. However, these proteins are distinct in their post-translational modifications, resulting in disparate cell trafficking routes and subcellular localization. Hence, HRAS, KRAS, and NRAS affect cellular processes in distinct ways. For example, HRAS is the most effective RAS protein at transforming fibroblasts. Furthermore, NRAS transforms hematopoietic cells most efficiently. Likewise, KRAS-deficient mice are embryonic lethal whereas NRAS or HRAS knock outs are essentially phenotypically normal (Parikh, et al., 2007).

[0042] The term "sites", and grammatical variations thereof, means any region of a protein, including those regions comprising the exterior, solvent-exposed portion of a protein. Such a site may be a pocket where other protein species or compounds interact with the RAS protein. Sites also may become available for binding upon conformation change. Compounds of the present invention may bind a RAS protein at two or more sites, including 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites on the RAS protein.

[0043] In one aspect of this embodiment, the compound selectively binds to another site on the RAS protein that comprises at least one amino acid from the switch I region (near D38). As used herein, "near", as it relates to distances from certain residues, such as D38, A59, L56, or 121 , means within about 9 angstroms of the residue, including, but not limited to, within 1 , 2, 3, 4, 5, 6, 7, or 8 angstroms of the residue on the RAS protein that corresponds to the amino acid number (such as 38, 59, 56, or 21 ) of the human HRAS protein. The corresponding regions of HRAS from other animal, as well as NRAS, KRAS, or other RAS proteins from human and other animals, are also within the scope of the present invention and are readily determined by one skilled in the art. See, e.g., Valencia et al., 1991 . "Corresponds," with reference to amino acid numbers on RAS, means consistent with, as done by sequence alignment. Multiple sequence alignment methods including pair-wise sequence alignment methods, may be used to determine the position in a RAS protein that corresponds to the positions listed above. Methods of sequence alignment are well-known. Many sequence alignment software programs are available. These programs include, e.g., BLAST, ClustalW, SEQALN, DNA Baser, MEME/MAST, BLOCKS, and eMOTIF. Preferably, the sequence alignment software is BLAST.

[0044] In some embodiments, the compound selectively binds to another site on the RAS protein that comprises at least one amino acid located between the switch 1 and switch 2 regions (near A59). [0045] In the present invention, the switch I region, located near the D38 site, includes residues 30-40 corresponding to the human RAS protein. The D38 site is one region of conserved interaction between RAS proteins and RAF, RALGDS, and PI3K. The switch II region is near A59 and comprises residues 60-70 corresponding to the human RAS protein. The A59 site is located between the switch I and switch II regions and is adjacent to the D38 site.

[0046] As used herein, "at least one amino acid" from any of the regions or locations of a RAS protein disclosed herein include 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids, up to, and including, the number of amino acids comprising the entire designated region or location of RAS.

[0047] In a further aspect of this embodiment, the RAS protein is an isoform selected from a group consisting of HRAS, KRAS, NRAS, and combinations thereof.

[0048] In an additional aspect of this embodiment, the RAS protein is an oncogenic mutant. As used herein, an "oncogenic mutant" is a RAS variant that contains an alteration in the amino acid sequence and has the potential to cause a cell to become cancerous. In the context of a RAS protein, an oncogenic mutant may be a constitutively active, continually GTP-bound isoform of RAS protein. Preferably, the RAS protein is an oncogenic mutant selected from a group consisting of HRAS ^G12D , KRAS ^G12D , NRAS ^{Q61 K} , NRAS ^G13V , and NRAS ^G13D , the mutations based on the human isoform for the respective protein. In terms of oncogenicity, mutations at residues 12-13 of a RAS protein render RAS's GTPase portion insensitive to activation by GAPs, while mutations at residue 61 affect the enzymatic active site of a RAS protein directly, thereby essentially inactivating the GTPase activity of a RAS protein. [0049] In another aspect of this embodiment, the compound selectively binds to at least one amino acid near L56, D38, and A59 in a RAS protein. Preferably, the compounds of the present invention comprise a region A that binds to at least one amino acid near D38 on a RAS protein and also comprise a heterocycle with at least one ring nitrogen.

[0050] As used herein, the term "heterocycle" means substituted or unsubstituted non aromatic ring structures. Preferably the heterocycle comprises 3 to 8 membered rings, and at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. Such heterocycles may include at least one ring nitrogen. The term "heterocycle" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic ring(s) can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocycle groups of the present invention include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0051] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur; more preferably, nitrogen and oxygen.

[0052] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by wherein R ⁷ , R ⁸ , and R ⁸ each independently represent a hydrogen or a hydrocarbyl group, or R ⁷ and R ⁸ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure. The term "primary" amine means only one of R ⁷ and R ⁸ or one of R ⁷ , R ⁸ , and R ⁸ is a hydrocarbyl group. Secondary amines have two hydrocarbyl groups bound to N. In tertiary amines, all three groups, R ⁷ , R ⁸ , and R ⁸ , are replaced by hydrocarbyl groups.

[0053] The term "C _x-y " when used in conjunction with a chemical moiety, such as, alkyl, alkenyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. For example, the term "Cx _-y alkyl" means substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc. The terms "C2- _y alkenyl" and "C2 _-y alkynyl" refer to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

[0054] The term "aliphatic", as used herein, means a group composed of carbon and hydrogen atoms that does not contain aromatic rings. Accordingly, aliphatic groups include alkyl, alkenyl, alkynyl, and carbocyclyl groups. A preferred Ci _-4 aliphatic is a vinyl moiety.

[0055] The term "alkyl" means the radical of saturated aliphatic groups that does not have a ring structure, including straight-chain alkyl groups, and branched-chain alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has 4 or fewer carbon atoms in its backbone (e.g., Ci-C ₄ for straight chains, C3-C ₄ for branched chains). [0056] The term "alkenyl", as used herein, means an aliphatic group containing at least one double bond.

[0057] The term "alkynyl", as used herein, means an aliphatic group containing at least one triple bond.

[0058] The term "aryl" as used herein includes substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 3- to 8-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g. , the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0059] The term "substituted" means moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with the permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g. , which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. The permissible substituents can be one or more and the same or different for appropriate organic compounds.

[0060] As used herein, a "halide" means a halogen atom such as fluorine, chlorine, bromine, iodine, or astatine.

[0061] As used herein, an "aromatic ring" is an aryl or a heteroaryl. The term "heteroaryl" includes substituted or unsubstituted aromatic single ring structures, preferably 3- to 8-membered rings, more preferably 5- to 7-membered rings, even more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The term "heteroaryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g. , the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

[0062] In a preferred embodiment, the A region of the compounds of the present invention comprise a fragment having formula (I):

wherein ring A is a heterocycle with at least one ring nitrogen, and Ri is selected from a group consisting of no atom, amine, and Ci _-4 aliphatic.

[0063] In another preferred embodiment, the A region of the compounds of the present invention is selected from a group consisting of:

[0064] In another preferred embodiment, the compounds of the present invention comprise a region B that binds to at least one amino acid near A59 on the RAS protein and comprises an indole. The term "indole" is art-recognized and means any compound containing a benzene ring fused to a pyrrole ring.

[0065] In a preferred aspect of this embodiment the B region of the compounds of the present invention comprise a structure of formula (III):

wherein

R ₃ is selected from a group consisting of heterocycle, aryl, and amine, which heterocycle, aryl, and amine may be optionally substituted with a group selected from halide, Ci _-4 aliphatic, and combinations thereof; and

R ₄ is selected from a group consisting of no atom, H, aryl, halide, Ci _-4 aliphatic, and -0-Ci _-4 aliphatic, wherein the Ci _-4 aliphatic is optionally substituted with halide. [0066] In a preferred aspect of this embodiment the B region of the compounds of the present invention is selected from a group consisting of

wherein

R ₂ and R ₅ are independently selected from a group consisting of no atom, aryl, and Ci _-4 aliphatic; and

R ₄ is selected from a group consisting of no atom, H, aryl, halide, Ci _-4 aliphatic, and -0-Ci _-4 aliphatic, wherein the Ci _-4 aliphatic is optionally substituted with halide.

[0067] In another embodiment of the present invention the compounds of the present invention comprise a region C, which region comprises a structure of formula (IV):

(IV)

wherein R ₆ is selected from a group consisting of no atom, H, alkyl, and aryl, wherein the alkyl is optionally substituted with a group consisting of halide, ether, and combinations thereof, and the aryl is optionally substituted with one or more groups consisting of halide, ether, Ci _-4 alkyl, and combinations thereof.

[0068] The term "ether", as used herein, means a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O- Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O- heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which may be represented by the general formula alkyl-O-alkyl.

[0069] In another embodiment of the present invention the compounds of the present invention comprise a region D that binds to at least one amino acid near L56 on the RAS protein comprising a structure of formula (VII):

wherein Ri ₂ , R13, Ri ₄ , and R15 are independently selected from a group consisting of H and -X-E, wherein X is selected from a group consisting of a bond, NH, 0, and S, and wherein E is an electrophile.

[0070] In the present invention, "electrophile" has its ordinary art-recognized meaning. Preferred electrophiles according to the present invention include:

wherein:

Y is selected from a group consisting of CI, Br, F, I, and N ₂ ;

Z is selected from a group consisting of O and NH; and

R-I 6 is selected from a group consisting of no atom, H, alkyl, and aryl, wherein the alkyl is optionally substituted with a group consisting of halide, ether, and combinations thereof, and the aryl is optionally substituted with a group consisting of halide, ether, Ci _-4 alkyl, and combinations thereof; and

p is 1 , 2, or 3.

[0071] Another embodiment of the present invention is a compound having the structure of formula (V):

R ₄ is selected from a group consisting of no atom, H, aryl, halide, Ci _-4 aliphatic, and -0-Ci _-4 aliphatic, wherein the Ci _-4 aliphatic is optionally substituted with one or more halide;

R ₈ is selected from a group consisting of no atom, H, alkyl, and aryl, wherein the alkyl is optionally substituted with a group consisting of halide, ether, and combinations thereof, and the aryl is optionally substituted with one or more groups consisting of halide, ether, Ci _-4 alkyl, and combinations thereof; R ₉ is selected from a group consisting of no atom, H, and aryl optionally substituted with a group consisting of ether, halide, and combinations thereof;

Ri2, Ri3, Ri4, and Ri ₅ are independently selected from a group consisting of H and -X-E, wherein X is selected from a group consisting of a bond, NH and 0, and wherein E is an electrophile;

m and n are independently selected from integers between 0-5; and ring A is a heterocycle with at least 1 ring nitrogen and optionally substituted with Ci _-4 alkyl or a halide,

or a crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0072] In the present invention, the term "crystalline form" means the crystal structure of a compound. A compound may exist in one or more crystalline forms, which may have different structural, physical, pharmacological, or chemical characteristics. Different crystalline forms may be obtained using variations in nucleation, growth kinetics, agglomeration, and breakage. Nucleation results when the phase-transition energy barrier is overcome, thereby allowing a particle to form from a supersaturated solution. Crystal growth is the enlargement of crystal particles caused by deposition of the chemical compound on an existing surface of the crystal. The relative rate of nucleation and growth determine the size distribution of the crystals that are formed. The thermodynamic driving force for both nucleation and growth is supersaturation, which is defined as the deviation from thermodynamic equilibrium. Agglomeration is the formation of larger particles through two or more particles (e.g. , crystals) sticking together and forming a larger crystalline structure. [0073] The term "hydrates", as used herein, means a solid or a semi-solid form of a chemical compound containing water in a molecular complex. The water is generally in a stoichiometric amount with respect to the chemical compound.

[0074] As used herein, "pharmaceutically acceptable salts" refer to derivatives of the compounds disclosed herein wherein the compounds are modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. For example, such salts include salts from ammonia, L-arginine, betaine, benethamine, benzathine, calcium hydroxide, choline, deanol, diethanolamine (2,2'- iminobis(ethanol)), diethylamine, 2-(diethylamino)-ethanol, 2-aminoethanol, ethylenediamine, N-ethyl-glucamine, hydrabamine, 1 H-imidazole, lysine, magnesium hydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassium hydroxide, 1 -(2- hydroxy-ethyl)-pyrrolidine, sodium hydroxide, triethanolamine (2, 2', 2"- nitrilotris(ethanol)), trometh-amine, zinc hydroxide, acetic acid, 2.2-dichloro-acetic acid, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 2,5-dihydroxybenzoic acid, 4-acetamido-benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, decanoic acid, dodecylsulfuric acid, ethane-1 ,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, ethylenediamonotetraacetic acid, formic acid, fumaric acid, galacaric acid, gentisic acid, D-glucoheptonic acid, D-gluconic acid, D- glucuronic acid, glutamic acid, glutantic acid, glutaric acid, 2-oxo-glutaric acid, glycero-phosphoric acid, glycine, glycolic acid, hexanoic acid, hippuric acid, hydrobromic acid, hydrochloric acid isobutyric acid, DL-lactic acid, lactobionic acid, lauric acid, lysine, maleic acid, (-)-L-malic acid, malonic acid, DL-mandelic acid, methanesulfonic acid, galactaric acid, naphthalene-1 ,5-disulfonic acid, naphthalene- 2-sulfonic acid, 1 -hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, octanoic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid (embonic acid), phosphoric acid, propionic acid, (-)-L-pyroglutamic acid, salicylic acid, 4-amino- salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)- L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid and undecylenic acid. Further pharmaceutically acceptable salts can be formed with cations from metals like aluminum, calcium, lithium, magnesium, potassium, sodium, zinc and the like, (also see Pharmaceutical salts, Berge, S.M. et al. , J. Pharm. Sci., (1977), 66, 1 -19).

[0075] The pharmaceutically acceptable salts of the present invention can be synthesized from a compound disclosed herein which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a sufficient amount of the appropriate base or acid in water or in an organic diluent like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, or a mixture thereof.

[0076] Another embodiment of the present invention is a compound having the structure of formula (VI):

wherein:

R ₄ is selected from a group consisting of no atom, H, aryl, halide, Ci _-4 aliphatic, and -0-Ci _-4 aliphatic, wherein the Ci _-4 aliphatic is optionally substituted with one or more halide;

R ₈ and R-n are independently selected from a group consisting of no atom, H, alkyi, and aryl, wherein the alkyi is optionally substituted with a group consisting of halide, ether, and combinations thereof, and the aryl is optionally substituted with one or more groups consisting of halide, ether, d. 4alkyl, and combinations thereof;

R-i o is selected from a group consisting of no atom, H, halide, and Ci _-4 aliphatic;

Ri2, Ri 3, Ri4, and R ₅ are independently selected from a group consisting of H and -X-E, wherein X is selected from a group consisting of a bond, NH and 0, and wherein E is an electrophile;

m and n are independently selected from integers between 0-5; and ring A is a heterocycle with at least 1 ring nitrogen and optionally substituted with Ci _-4 alkyl or a halide,

or a crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0077] As used herein, an "integer between 0-5" means 0, 1 , 2, 3, 4, or 5.

[0078] Examples of preferred compounds of the present invention include:

and crystalline forms, hydrates, or pharmaceutically acceptable salts thereof.

[0079] Preferably, each compound of the present invention defined by or including a fragment defined by formulae I - VII selectively binds a RAS protein at three or more sites. More preferably such compounds selectively bind to a site on the RAS protein that comprises at least one amino acid near L56, a site on the RAS protein that comprises at least one amino acid from the switch 2 region (near D38), and a site on the RAS protein that comprises at least one amino acid between the switch 1 and switch 2 regions (near A59). The compounds of the present invention may selectively bind to 4 or more sites on the RAS protein such as 5, 6, 7, 8, 9, or 10 sites.

[0080] Another embodiment of the present invention is a compound having

the structure: wherein Ri ₂ , R13, Ri ₄ , and R-15 are independently selected from a group consisting of H and -X-E, wherein X is selected from a group consisting of a bond, NH and 0, and wherein E is an electrophile,

or a crystalline form, hydrate, or pharmaceutically acceptable salt thereof,

with the provisio that at least one of R-12, R13, Ri ₄ , and R-1 5 are not H.

[0081] Another embodiment of the present invention is a compound selected from a group consisting of

and crystalline forms, hydrates, or pharmaceutically acceptable salts thereof.

[0082] Another embodiment of the present invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more compounds of the present invention.

[0083] The compounds or compositions, including pharmaceutical compositions, of the present invention may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, compounds or compositions, including pharmaceutical compositions, of the present invention may be administered in conjunction with other treatments. Compounds or compositions, including pharmaceutical compositions, of the present invention may be encapsulated or otherwise protected against gastric or other secretions, if desired.

[0084] The compositions, including pharmaceutical compositions, of the invention comprise one or more active ingredients in admixture with one or more pharmaceutically-acceptable diluents or carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the active agents/compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g. , Remington, The Science and Practice of Pharmacy (21 ^st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.).

[0085] Pharmaceutically acceptable diluents or carriers are well known in the art (see, e.g. , Remington, The Science and Practice of Pharmacy (21 ^st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g. , lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g. , dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g. , saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g. , ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g. , glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g. , ethyl oleate and tryglycerides), biodegradable polymers (e.g. , polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g. , corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g. , suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable diluent or carrier used in a pharmaceutical composition of the invention must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Diluents or carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable diluents or carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.

[0086] The compositions, including pharmaceutical compositions, of the invention may, optionally, contain additional ingredients and/or materials commonly used in pharmaceutical compositions. These ingredients and materials are well known in the art and include (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (1 1 ) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface-active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21 ) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.

[0087] The compositions, including pharmaceutical compositions, of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.

[0088] Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable diluents or carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form. [0089] Liquid dosage forms for oral administration include pharmaceutically- acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.

[0090] The compositions, including pharmaceutical compositions, of the present invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating diluents or carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. The pharmaceutical compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable diluents or carriers as are known in the art to be appropriate.

[0091] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable diluent or carrier. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.

[0092] The compositions, including pharmaceutical compositions, of the present invention suitable for parenteral administrations may comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These pharmaceutical compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.

[0093] In some cases, in order to prolong the effect of a drug (e.g., pharmaceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.

[0094] The rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-adm inistered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.

[0095] Any formulation of the invention may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid diluent or carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

[0096] An additional embodiment of the present invention is a method for treating or ameliorating the effects of a disease associated with altered RAS signaling in a subject. The method comprises administering to the subject an effective amount of any compound disclosed herein.

[0097] In the present invention, an "effective amount" or a "therapeutically effective amount" of a compound or composition disclosed herein is an amount of such compound or composition that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of mammal, e.g., human patient, and like factors well known in the arts of medicine and veterinary medicine. In general, a suitable dose of a compound or composition, including pharmaceutical compositions, according to the invention will be that amount of the compound or composition which is the lowest dose effective to produce the desired effect. The effective dose of a compound or composition, including pharmaceutical compositions, of the present invention may be administered as two, three, four, five, six or more sub- doses, administered separately at appropriate intervals throughout the day.

[0098] A suitable, non-limiting example of a dosage of any of the compounds or compositions, including pharmaceutical compositions, disclosed herein is from about 1 mg/kg to about 2400 mg/kg per day, such as from about 1 mg/kg to about 1200 mg/kg per day, 75 mg/kg per day to about 300 mg/kg per day, including from about 1 mg/kg to about 100 mg/kg per day. Other representative dosages of such agents include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1 100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg, and 2300 mg/kg per day. The effective dose of compounds or compositions, including pharmaceutical compositions, disclosed herein, may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.

[0099] As used herein, the terms "ameliorate", "ameliorating" and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject.

[0100] As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g. , a patient. In particular, the methods and compositions, including pharmaceutical compositions, of the present invention may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.

[0101] As used herein, a "subject" is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present invention include, for example, primates, farm animals, domestic animals, laboratory animals, etc. Some examples of agricultural animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc.

[0102] As used herein, the phrase "altered RAS signaling" means any deviation in the activity of a RAS protein from that typically observed from wild-type RAS protein in a given tissue. Altered RAS signaling may include, for example, increased RAS signaling or decreased RAS signaling. Altered RAS signaling may be caused by one or more mutations in the RAS protein, such as the oncogenic mutations disclosed above. For example, certain RAS protein mutations may enable RAS protein to constitutively exist in its GTP-bound conformation, either by discouraging interaction of RAS protein with various GAP proteins or by disabling the GTPase activity of RAS protein. [0103] As used herein, a RAS protein includes, but is not limited to any of the members of the RAS subfamily. Non-limiting examples of RAS subfamily members are set forth in Table 1 , below.

Table 1 : Example RAS Subfamily Members

Protein Synonym Accession

Number

Ras Subfamily NP_005334

H-Ras, isoform 1 NP_789765

H-Ras, isoform 2 H-RaslDX NP_002515

N-Ras NP_004976

K-Ras2B NP 203524

NP 006261

K-Ras2A NP 036382

NP 036351

R-Ras NP 002875

NP 056461

TC21 R-Ras2 NP 066361

NP_002877

M-Ras R-Ras3

NP 067006

RaplA Krev-1/Smgp21 NP_008843

NP 002921

Rapl B NP 054731

AAH35663

Rap2A NP 004156

NP 859053

Rap2B NP 005605

NP 653194

Rap2C NP 004666

NP 660156

Rit1 Roc1/RibB Rin/Roc2/RibA NP 060064

NP 853510

Rit2 Ges

NP 1 16307

NP 005393

Rem1

AAA36542

Rem2 R-Rad/Rem3 NP 002872

NP 065078

Rad Kir NP 060065

NP 057168

Gem Rheb2 NP 055125

NP 006468

NP 201572 Protein Synonym Accession

Number

Rhebl RhebU NP 996563

NP 076429

Rheb2 ARHI/Rhol Rig/GBTS1 NP_057647

Noey2 NP_079006

Di-Ras1 H-Ras2/H-RasP

Di-Ras2

E-Ras

Rerg

RalA, isoform 1

RalA, isoform 2 κΒ-Rasl

RalB KB-Ras2

NKIRasl DexRas/Ags1 Rhes/Tem2

NKIRas2 RasLI OA

RasD1

RasD2

RRP22

RasLI OB

RasL1 1A

RasL1 1 B

Ris/RasL12

[0104] In the present invention, a disease associated with altered RAS signaling may be a cancer, a neurological disorder, a metabolic disorder, an immunological disorder, an inflammatory disorder, and a developmental disorder. Preferably, the disease is autism, rasopathies, neurofibromatosis type 1 , Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome, hereditary gingival fibromatosis type 1 , Legius syndrome, Leopard syndrome, diabetic retinopathy, diabetes, hyperinsulinemia, chronic idiopathic urticarial, autoimmune lymphoproliferative syndrome, and capillary malformation-arteriovenous malformation.

[0105] Another embodiment of the present invention is a method for treating or ameliorating the effects of a disease associated with altered RAS signaling in a subject comprising administering to the subject an effective amount of a pharmaceutical composition of the present invention.

[0106] Another embodiment of the present invention is a method for effecting cancer cell death comprising contacting a cancer cell with an effective amount of a compound of the present invention. In this embodiment, "contacting" means bringing the compound into close proximity to the cancer cell. This may be accomplished using conventional techniques of drug delivery to mammals or in the in vitro situation by, e.g., providing the compound to a culture media in which the cancer cell is located.

[0107] Suitable and preferred compounds are as disclosed herein. In this embodiment, effecting cancer cell death may be accomplished in cancer cells having various mutational backgrounds as disclosed above.

[0108] The methods of this embodiment, which may be carried out in vitro or in vivo, may be used to effect cancer cell death by, e.g., killing cancer cells, in cells of the types of cancer disclosed herein.

[0109] In one aspect of this embodiment, the cancer cell is a mammalian cancer cell. Preferably, the mammalian cancer cell is obtained from a mammal selected from a group consisting of humans, primates, farm animals, and domestic animals and laboratory animals. More preferably, the mammalian cancer cell is a human cancer cell.

[0110] In the present invention, cancers include both solid and hemotologic cancers. Non-limiting examples of solid cancers include adrenocortical carcinoma, anal cancer, bladder cancer, bone cancer (such as osteosarcoma), brain cancer, breast cancer, carcinoid cancer, carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing family of cancers, extracranial germ cell cancer, eye cancer, gallbladder cancer, gastric cancer, germ cell tumor, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer, large intestine cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, malignant mesothelioma, Merkel cell carcinoma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell cancer, pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, transitional cell cancer of the renal pelvis and ureter, salivary gland cancer, Sezary syndrome, skin cancers (such as cutaneous t-cell lymphoma, Kaposi's sarcoma, mast cell tumor, and melanoma), small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0111] Examples of hematologic cancers include, but are not limited to, leukemias, such as adult/childhood acute lymphoblastic leukemia, adult/childhood acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia, lymphomas, such as AIDS-related lymphoma, cutaneous T-cell lymphoma, adult/childhood Hodgkin lymphoma, mycosis fungoides, adult/childhood non-Hodgkin lymphoma, primary central nervous system lymphoma, Sezary syndrome, cutaneous T-cell lymphoma, and Waldenstrom macroglobulinemia, as well as other proliferative disorders such as chronic myeloproliferative disorders, Langerhans cell histiocytosis, multiple myeloma/plasma cell neoplasm, myelodysplastic syndromes, and myelodysplastic/myeloproliferative neoplasms.

[0112] Preferably, the cancer is selected from a group consisting of pancreatic cancer, colorectal cancer, lung cancer, skin cancer, urinary bladder cancer, thyroid cancer, hematopoietic cancer, prostate cancer, breast cancer, liver cancer, soft tissue cancer, leukemia and bone cancer.

[0113] In a preferred aspect of this embodiment, the cancer is selected from a group consisting of pancreatic cancer, colorectal cancer, fibrosarcoma, breast cancer, lung cancer, skin cancer, leukemia and bone cancer.

[0114] Another embodiment of the present invention is a kit for treating or ameliorating the effects of a disease related to altered RAS signaling in a subject in need thereof, the kit comprising an effective amount of a compound or pharmaceutical composition of the present invention packaged together with instructions for its use.

[0115] Another embodiment of the present invention is a kit for treating or ameliorating the effects of a cancer in a subject in need thereof, the kit comprising an effective amount of a compound or pharmaceutical composition of the present invention, packaged together with instructions for its use. [0116] The kits of the present invention may also include suitable storage containers, e.g. , ampules, vials, tubes, etc., for the compounds and compositions of the present invention and other reagents, e.g. , buffers, balanced salt solutions, etc., for use in administering the compounds and compositions to subjects. The compounds and compositions, including pharmaceutical compositions, of the present invention may be present in the kits in any convenient form, such as, e.g. , in a solution or in a powder form. The kits may further include a packaging container, optionally having one or more partitions for housing the compounds and pharmaceutical compositions and other optional reagents.

[0117] Another embodiment of the present invention is a composition comprising a compound of the present invention.

[0118] In one aspect of this embodiment, the composition is a research reagent. As used herein, a "research reagent" is any compound or composition used in the execution of investigational activities.

[0119] Another embodiment of the present invention is a compound that selectively binds a RAS protein at three or more sites, wherein when the compound is bound to the RAS protein, the compound binds to L56 of the RAS protein and blocks the binding of SOS to the RAS protein.

[0120] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0121] For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.

EXAMPLES

[0122] The following examples are provided to further illustrate certain aspects of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.

Example 1

Experimental Data

[0123] Software. Molecular docking was performed using GLIDE (Schrodinger, Inc). Modeling of proteins and ligands were performed using Molecular Operating Environment [MOE] (Chemical Computing Group).

[0124] In silico libraries. Libraries of commercially available compounds were compiled from the inventories of Asinex, Enamine, Chembridge, ChemDiv, IBS, Life, Maybridge and TimTec. A fragment subset of ~60,000 compounds of the unfiltered library was selected using the following filter criteria: LogP < 3, hydrogen bond acceptors <= 3, hydrogen bond donors <= 3, molecular weight < 300, aqueous solubility > 0.5 mM. Chemical descriptors were calculated using MOE (Chemical Computing Group).

[0125] Mic rosea le thermophoresis GTP-loaded KRAS ^G12D was labeled at a surface-exposed cysteine residue using a NT-647-maleimide dye. Thermophoretic movement of the fluorescently labeled protein with the inhibitors was measured in capillaries across a 16-point dilution series using a Monolith NT.1 15 (Nanotemper Technologies). [0126] General Procedures. All reactions were carried out under a nitrogen atmosphere under anhydrous conditions unless indicated otherwise. Anhydrous methylene chloride (CH ₂ CI ₂ ) and tetrahydrofuran (THF) were purchased from Sigma- Aldrich. Reactions were magnetically stirred and monitored by thin layer chromatography on Merck pre-coated 0.25 mm silica plates containing a 254 nm fluorescence indicator. Flash chromatography was performed on a Teledyne combiflash companion automatic flash chromatography system. Preparative thin layer chromatography was performed on 1 mm SiliCycle prep plates. NMR spectra were obtained on a Bruker DPX 300 or 400 MHz spectrometer.

[0127] Abbreviations. EDIPA = diisopropylethyl amine, EtOAc = ethyl acetate, MeOH = methanol, DCE = 1 ,2-dichloroethane, Na ₂ S0 ₄ = sodium sulfate, NaHC0 ₃ = sodium bicarbonate, Pd(PPh ₃ ) ₄

Tetrakis(triphenylphosphine)palladium(0), NH ₄ CI = ammonium chloride, TFA = trifluoroacetic acid, HBTU = 0-(Benzotriazol-1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, HCI = hydrochloric acid, THF = tetrahydrofuran, K2CO3 = potassium carbonate. CH ₂ CI ₂ = Dichloromethane, EtOH = ethanol.

[0128] Boc-amino Indole S1. To a solution of 1 H-indole-5-carbaldehyde (3.5 g, 24 mmol, 1 .0 equiv) in DMF (100 mL) at 0°C, sodium hydride (60% in mineral oil) (1.1 g, 28.8 mmol, 1 .2 equiv) was added in several portions over 5 minutes. The mixture was stirred for 45 minutes at 0°C before the sequential addition of 3-(Boc- amino)propyl bromide (8.00 g, 33.6 mmol, 1.4 equiv) and sodium iodide (3.6 g, 24 mmol, 1 .0 eq). The solution was warmed to 80°C and stirred for 48 hours. Upon completion, the reaction was diluted with saturated aqueous NaHCOs and extracted 3 times with EtOAc. The combined organic layers were washed with brine, dried (Na ₂ S0 ₄ ), concentrated, and the crude material was purified by flash column chromatography (silica gel, hexanes/EtOAc, 1 : 1 ) (3.4 g, 47% yield). ¹ H NMR (400 MHz, CDCIs) δ 10.05 (s, 1 H), 8.18 (d, J = 1.5 Hz, 1 H), 7.81 (dd, J = 8.7, 1 .6 Hz, 1 H), 7.26 (d, J = 3.1 Hz, 1 H), 6.75-6.60 (m, 1 H), 4.54 (s, 1 H), 4.25 (t, J = 6.9 Hz, 2H), 3.17 (d, J = 7.2 Hz, 2H), 2.08 (p, J = 6.9 Hz, 2H), 1 .47 (s, 8H). ¹³ C NMR (101 MHz, CDCI3) 192.44, 156.08, 139.19, 129.79, 129.34, 128.41 , 126.56, 121 .80, 109.80, 103.60, 44.05, 38.04, 30.63, 28.38. HRMS (m/z): [M ⁺ ] calc'd for C17H22N2O3, 302.37, found 302.16.

[0129] Bromide 9 To a solution of S1 (FIG. 8) (1 .8 g, 5.95 mmol, 1 .0 equiv) in THF (120 mL) at -78°C, bromine (0.367 mL, 7.1 mmol, 1 .2 equiv) was added dropwise over 5 minutes. The resulting mixture was stirred at -78°C for 2 hours. Upon completion, the reaction contents were poured onto a solution of ice (~300 g), water (200 mL), ammonium hydroxide (1 mL, 12 M), sodium thiosultate pentahydrate (1 mL, saturated solution in water). The crude material was extracted with EtOAc (3 x 30 mL) and the combined organic extracts were then washed with water (100 mL) and brine (100 mL), dried (Na ₂ S0 ₄ ), filtered, and concentrated. The crude material was purified by flash column chromatography (silica gel, hexanes/EtOAc, 1 :1 ) to afford 9 (1.2 g, 53% yield). 9: ¹ H NMR (400 MHz, CDCI ₃ ) δ 10.09 (s, 1 H), 8.12 (d, J = 1 .5 Hz, 1 H), 7.86 (dd, J = 8.7, 1 .5 Hz, 1 H), 7.43 (d, J = 8.6 Hz, 1 H), 7.30 (s, 1 H), 4.57 (s, 1 H), 4.23 (t, J = 6.9 Hz, 2H), 3.18 (d, J = 6.8 Hz, 2H), 2.08 (q, J = 6.7 Hz, 2H), 1 .47 (s, 9H). ¹³ C NMR (101 MHz, CDCI ₃ ) 191 .19, 155.31 , 137.89, 128.98, 127.87, 126.53, 124.05, 121 .63, 109.40, 91 .20, 78.67, 76.65, 76.33, 76.01 , 43.54, 37.09, 29.82, 27.57, 27.54. HRMS (m/z): [M ⁺ ] calc'd for Ci7H ₂ i BrN ₂ 03, 381 .26, found 380.07. (FIG. 6)

[0130] Benzyl Alcohol 10 To a solution of 9 (1 .60 g, 4.20 mmol, 1 .0 equiv) in dioxane (10 mL), 3-(hydroxylmethyl)phenylboronic acid (0.640 g, 4.20 mmol, 1 .0 eq), Pd(PPh ₃ ) ₄ (0.250 g, 0.195 mmol, 0.05 equiv), and a solution of K ₂ C0 ₃ (1 .10 g, 8.40 mmol, 2.0 equiv) in water were added sequentially. The resulting mixture was heated to 80°C and stirred for 48 hours. Upon completion, the reaction was diluted with saturated aqueous NaHCOs and extracted with EtOAc (3 x 10 mL). The combined organic extracts were then washed with water (20 mL) and brine (20 mL), dried (Na ₂ S0 ₄ ), filtered, and concentrated. The crude material was purified by flash column chromatography (silica gel, hexanes/EtOAc, 1 : 1 ) to afford 10 (1 .4 g, 85% yield). ¹ H NMR (400 MHz, CDCI ₃ ) δ 10.1 1 (s, 1 H), 8.41 (s, 1 H), 7.85 (dd, J = 1.1 Hz, 8.6 Hz, 1 H), 7.65 (m, 2 H), 7.47 (m, 3 H), 7.37 (d, J = 7.6 Hz, 1 H), 7.29 (s, 1 H), 4.70 (s, 3 H), 4.29 (t, J = 7.0 Hz, 2 H), 3.21 (m, 2 H), 2.12 (t, J = 6.8 Hz, 2 H), 1 .46 (s, 9 H). (FIG. 6)

[0131] Aldehyde 12 To a solution of 10 (0.250 g, 0.61 mmol, 1 .0 equiv) in CH ₂ CI ₂ (3 mL) at 0°C was added triethylamine (0.2 mL, 1 .22 mmol, 2.0 equiv) and methanesulfonyl chloride (0.1 mL, 1 .22 mmol, 2.0 equiv). The reaction was allowed to warm to 25°C and stirred for 2 hours. Upon completion, the reaction was quenched with saturated aqueous NH ₄ CI and extracted with CH2CI2 (3 x 5 mL). The combined organic extracts were then washed with water (20 mL) and brine (20 mL), dried (Na ₂ S0 ₄ ), filtered, and concentrated. A portion of the crude material 11 (0.130 g, 0.27 mmol, 1 .0 equiv) was taken forward without further purification, and dissolved in DMF (2 mL). Indole L56 fragment (0.070 g, 0.27 mmol, 1 .0 equiv, prepared according to a published procedure from Sun et al. Ang. Chem. Int. Ed. 2012, 51 , 6140), was added as a solution in DMF (1 mL). Triethylamine (0.1 1 mL, 0.81 mmol, 3.0 equiv) and sodium iodide (0.120 g, 0.8 mmol, 3.0 equiv) were added sequentially, and the solution was heated at 80°C overnight. Upon completion, the reaction was quenched with saturated aqueous NH ₄ CI and extracted with CH2CI2 (3 x 5 mL). The combined organic extracts were then washed with water (20 mL) and brine (20 mL), dried (Na ₂ S0 ₄ ), filtered, and concentrated. The crude material was purified by flash column chromatography (silica gel, 10% MeOH in CH2CI2) to afford 12 (0.140 g, 80% yield). 12: ¹ H NMR (400 MHz, CDCI ₃ ) δ 9.69 (s, 1 H), 8.22 (s, 1 H), 7.76 (m. 2 H), 7.58-7.53 (m, 3 H), 7.43-7.29 (m, 5 H), 7.17 (s, 1 H), 7.07 (t, J = 7.1 Hz, 1 H), 6.92 (t, J = 7.2 Hz, 1 H), 6.74 (m, 2 H), 4.45 (s, 2 H), 4.28 (m, 4 H), 3.08 (m, 2 H), (2.01 (m, 3 H), 1 .45 (s, 9 H). (FIG. 6)

[0132] 1-(2,6-dichlorobenzyl)piperazine. To a solution of piperazine (1 12 mmol, 6.0 eq) in THF (180 mL) at 0°C, a solution of 2,6-dichlorobenzyl bromide (4.5 g, 18.8 mmol) in THF (20 mL) was added dropwise over 10 minutes. The resulting mixture was slowly allowed to warm to room temperature and stirred for 24 hours. Upon completion the THF was removed and the crude material was re-suspended in CH2CI2 and water, and extracted 2 additional times with CH2CI2. The combined organic layers were dried (Na ₂ S0 ₄ ), concentrated, and the crude material was purified by flash column chromatography (20% MeOH in CH ₂ CI ₂ ) to afford 1 -(2,6- dichlorobenzyl)piperazine (2.3 g, 50% yield). ¹ H NMR (400 MHz, Methanol-d ₄ ) δ 7.62 -7.30 (m, 2H), 7.23 (dd, J = 8.7, 7.4 Hz, 1 H), 3.74 (s, 2H), 2.92-2.69 (m, 4H), 2.56 (t, J = 4.9 Hz, 4H). ¹³ C NMR (101 MHz, MeOD) 136.76, 133.67, 129.18, 128.24, 56.55, 53.41 , 44.95. HRMS (m/z): [M ⁺ ] calc'd for Cn H ₁₄ CI ₂ N ₂ , 245.15, found 245.06.

[0133] Piperazine 13 To a solution of 12 (0.1 10 g, 0.17 mmol, 1 .0 equiv) in DCE (2 mL), 1 -(2,6-dichlorobenzyl)piperazine (0.125 g, 0.50 mmol, 3.0 equiv) and zinc chloride (5.0 mg, 0.034 mmol, 0.2 equiv) were added sequentially. The resulting mixture was stirred at 60°C for 2 hours before the addition of a solution of sodium cyanoborohydride (20.0 mg, 0.34 mmol, 2.0 equiv) in methanol (0.5 mL). The resulting mixture was stirred for an additional 2 hours at 60°C. Upon completion the reaction was concentrated and purified directly by flash column chromatography (silica gel, 10% MeOH in CH ₂ CI ₂ ) to afford 13 (0.090 g, 60% yield). 13: ¹ H NMR (400 MHz, MeOD) δ 7.73 (s, 1 H), 7.52 (s, 1 H), 7.47-7.07 (m, 14 H), 6.96 (m, 1 H), 6.71 (dd, J = 2.1 Hz, 8.7 Hz, 1 H), 6.66 (d, J = 1 .7 Hz, 1 H), 4.42 (s, 2 H), 4.27 (s, 2 H), 4.21 (s, 2 H), 3.73 (s, 2 H), 2.60 (br s, 4 H), 2.47 (br s, 4 H), 1 .99 (t, J = 6.8 Hz, 2 H), 1 .42 (s, 9 H). (FIG. 6)

[0134] 04SBT04 To a solution of 13 (0.100 g, 0.12 mmol, 1 .0 equiv) in dioxane (2 ml_), excess HCI (0.1 ml_, 4.0 M in dioxane) was added and the resulting solution was stirred for 24 hours. Upon completion, the dioxane was removed and the crude material was re-suspended in methanol and an excess of K2CO3 was added (~100 mg). The slurry was stirred at room temperature for 1 hour to ensure basification. K2CO3 was filtered off, and the solution was concentrated and purified by flash column chromatography (silica gel, 20% MeOH in CH2CI2) to afford S2. A portion of this material (0.025 g, 0.032 mmol, 1 .0 equiv) was added to a pre-stirred (0°C, 30 minutes) solution of HBTU (0.014 g, 0.05 mmol, 1 .5 equiv), 4-amino-1 -(tert- butoxycarbonyl)piperidine-4-carboxylic acid (0.012 g, 0.05 mmol, 1 .5 equiv) and EDIPA (0.01 ml_, 0.05 mmol, 2.0 equiv)) in DMF (2 ml_) and stirred for an additional 6 h at 25°C. Upon completion, the reaction was quenched with saturated aqueous NaHCO3 and extracted with EtOAc (3 x 2 ml_). The combined organic layers were washed with water (10 ml_), brine (10 ml_), dried (Na ₂ SO ₄ ), concentrated, and purified by flash column chromatography (silica gel, 20% MeOH in CH2CI2) to afford S3. A portion of this slightly impure material (0.020 g) was dissolved in dioxane (1 ml_) and HCI (0.1 ml_, 4.0 M in dioxane) was added. The resulting mixture was stirred for 6 h at 25°C. After removal of dioxane, the residue was re-suspended in MeOH and excess K2CO3 (100 mg) was added. After stirring for 1 h, K2CO3 was filtered off, and the solution was concentrated and purified by preparative TLC (20% MeOH in DCM) to afford 04SBT04 (0.010 g). 04SBT04: ¹ H NMR (400 MHz, MeOD) δ 7.76 (s, 1 H), 7.58 (s, 1 H), 7.52-7.17 (m, 13 H), 7.08 (t, J = 7.2 Hz, 1 H), 6.92 (t, J = 7.2 Hz, 1 H), 6.72 (m, 2 H), 4.46 (m, 3 H), 4.29 (m, 5 H), 3.77 (m, 3 H), 2.62 (br s, 4 H), 2.45 (br s, 4 H), 2.1 1 (m, 2 H), 1 .48 (m, 1 H). (FIGs. 6 and 8)

[0135] Acid 15 To a solution of 2-(5-nitro-1 H-indol-3-yl)acetic acid (14, 0.200 g, 0.92 mmol, 1 .0 equiv) in MeOH (5 mL), excess pTsOH (0.800 g, 4.5 mmol, 5.0 equiv) was added. The solution was heated under reflux for three days, quenched with saturated aqueous NaHCO ₃ , poured into water and extracted with EtOAc (3 x 5 mL). The combined organic extracts were then washed with water (10 mL) and brine (10 mL), dried (Na ₂ SO ₄ ), filtered, and concentrated to afford pure methyl ester S4 in quantitative yield. The crude material was reduced under hydrogen atmosphere in the presence of catalytic Pd/C in EtOAc overnight to afford S5. This material was advanced without further purification and the aryl amino group was Boc-protected with excess Boc ₂ O and NaHCO ₃ in THF for 2 h at 25°C to afford S6. Crude S6 was subsequently subjected to excess KOH in THF/water overnight in order to saponify the ester to the corresponding carboxylic acid 15 (0.180 g, 67% yield from 14). 15: ¹ H NMR (400 MHz, CDCI ₃ ) δ 8.34 (s, 1 H), 7.61 (s, 1 H), 7.18 (m, 2 H), 6.59 (s, 1 H),

3.75 (s, 2 H), 1 .56 (s, 9 H). (FIG. 7A and FIG. 8)

[0136] Amino-indole 18. Intermediate 15 (0.250 g, 0.86 mmol) was advanced towards 17 using the identical conditions previously described by Sun et al. 17 could be subsequently deprotected using excess HCI (4.0 M in dioxane) overnight to afford 18 (0.100 g, 38% yield from 15). 18: ¹ H NMR (400 MHz, CDCI ₃ ) δ 8.08 (d, J = 2.6 Hz, 1 H), 7.91 (dd, J = 2.6 Hz, 9.0 Hz, 1 H), 7.23 (m, 2 H), 7.08 (d, J = 1.8 Hz, 1 H),

6.76 (m, 2 H), 3.85 (s, 2 H). (FIG. 7A) [0137] Vinyl amide 19 To a solution of 18 (0.012 g, 0.04 mmol, 1 .0 equiv) in

THF (1 mL) was added excess triethylamine (0.01 mL) and acryloyl chloride (0.01 mL, 0.1 mmol, 2.5 equiv) at 0°C. After stirring at 0°C for 1 hour, the reaction contents were quenched with saturated aqueous NH ₄ CI, poured into water and extracted with CH ₂ CI ₂ (3 x 5 mL). The combined organic extracts were then washed with water (10 mL) and brine (10 mL), dried (Na ₂ S0 ₄ ), filtered, and concentrated. The resultant crude, yellow oil was purified by preparative TLC (10% MeOH in CH ₂ CI ₂ ) to afford S7 (0.010 g, 69% yield). This material was then reduced with catalytic Fe/HCI in a mixture of EtOH/water to afford 19 in quantitative yield. 19: ¹ H NMR (400 MHz, MeOD) δ 7.69 (s, 1 H), 7.37 (m, 2 H), 7.27 (d, J = 8.5 Hz, 1 H), 7.23 (s, 1 H), 6.85 (d, J = 1 .7 Hz, 1 H), 6.71 (dd, J = 2.1 Hz, 8.5 Hz, 1 H), 6.44 (dd, J = 1 10.0 Hz, 17.0 Hz, 1 H), 6.34 (dd, J = 1.7 Hz, 10.0 Hz, 1 H), 5.73 (dd, J = 1 .7 Hz, 17 Hz, 1 H), 4.32 (s, 2 H). (FIG. 7B and FIG. 8)

[0138] Chloroacetamide 20 To a solution of 18 (0.012 g, 0.04 mmol, 1 .0 equiv) in THF (1 mL) was added excess triethylamine (0.01 mL) and chloroacetyl chloride (0.01 mL, 0.1 mmol, 2.5 equiv) at 0°C. After stirring at 0°C for 1 hour, the reaction contents were quenched with saturated aqueous NH ₄ CI, poured into water and extracted with CH ₂ CI ₂ (3 x 5 mL). The combined organic extracts were then washed with water (10 mL), brine (10 mL), dried (Na ₂ SO ₄ ), filtered, and concentrated. The resultant crude, yellow oil was purified by preparative TLC (10% MeOH in CH ₂ CI ₂ ) to afford S8 (0.009 g, 57% yield). This material was then reduced under hydrogen atmosphere in the presence of catalytic Pd/C in EtOAc overnight to afford 20 in quantitative yield. 20: ¹ H NMR (400 MHz, MeOD) δ 7.74 (dd, J = 1 .5 Hz, 8.5 Hz, 1 H), 7.42-7.27 (m, 4 H), 7.18 (d, J = 2.5 Hz, 1 H), 6.83 (d, J = 8.5 Hz, 1 H), 6.52 (m, 1 H), 4.21 (s, 2 H), 3.85 (s, 2 H). (FIG. 7B and FIG. 8) [0139] Aldehyde 21. To a solution of 18 in THF excess triethylamine and 2- iodoethanol are added at 0°C. After stirring at 0°C, the reaction contents are quenched with saturated aqueous NH ₄ CI, poured into water and extracted with CH2CI2. The combined organic extracts are then washed with water and brine, dried (Na ₂ S0 ₄ ), filtered, and concentrated. The resultant crude, product is purified by preparative TLC. Oxidation of the primary alcohol to an aldehyde is accomplished by, for example, Swern oxidation. This material is then reduced under hydrogen atmosphere in the presence of catalytic Pd/C in EtOAc overnight to afford 21 (FIG. 7B).

Example 2

[0140] Targeting RAS PPIs initially relied upon analysis of the co-crystal structures of HRAS with ΡΙ3Κγ (PDB: 1 HE8), with the RAF RAS-binding domain (RBD, PDB: 3KUD), and with RALGDS (PDB: 1 LFD) in order to gain insights regarding conserved effector regions. A particularly persistent site of RAS-effector interaction sits along a short stretch of amino acids within the switch I region (red), highlighted in FIG. 3A to display the apparent overlap with the D38 site (green) shown in FIG. 3B. Subsequent analysis of the KRAS ^G12D (PDB: 4DSN) structure revealed a shallow pocket directly adjacent to the D38 site in between the switch I and switch II (blue) regions, denoted as the A59 site (purple). These observations informed a multivalent ligand approach targeting the D38 and A59 sites based on in silico library design and fragment screening, resulting in 31 MEW44, a novel inhibitor of KRAS ^G12D .

[0141] Design of Novel Three-Site Compounds. Further analysis of the proposed binding mode of 31 MEW44 with KRAS ^G12D divulged an additional shallow pocket adjacent to the trifluoromethylphenyl ring near L56 (FIG. 4A). This region has previously been the focus of several fragment-based screening efforts described earlier in this section, as it is the site of SOS-mediated nucleotide exchange towards activated GTP-bound KRAS (Sun et al., Maurer et al.). As such, it is a relevant effector region to GDP-bound RAS, while efforts have extensively focused on the GTP-bound conformation to this point. The possibility of extending into this pocket through derivation of 31 MEW44 at the 3-position of the aryl ring neighboring the L56 site would potentially generate compounds targeting three distinct sites to infer greater inhibitory efficiency and binding affinity for KRAS ^G12D .

[0142] Towards accomplishing this goal, an in silico screen of 60,000 commercially available fragments constrained to the L56 residue was conducted. The top-scoring fragment is pictured in FIG. 4B. While this particular substrate is not readily accessible through synthetic transformations that would facilitate incorporation into an analog of 31 MEW44, it shared an obvious resemblance to the synthetically feasible benzimidazole/indole-based scaffold (L56-fragment). This scaffold was the basis of inhibitors developed by Sun et al., which targeted an identical region of GDP-bound RAS. Virtual docking of a fully elaborated 3-site compound (L56 3-site) returned the anticipated docking pose (FIG. 5A) with the newly introduced fragment making multiple contacts at the L56 site, namely electrostatic interactions with Q37 and T74 (FIG. 5B). Intriguingly, the docking score of this novel 3-site compound was significantly higher than that of 31 MEW44 (docking score = -9.3), further propagating the notion that we could achieve augmented inhibition by simultaneously targeting an alternate site.

[0143] Synthesis of the target compound was accomplished as outlined in Scheme 1 (FIG. 6). Alterations to the original 31 MEW44 route, specifically the exchange of the trifluoromethyl ether aryl boronic acid to a benzyl alcohol analog, afforded the opportunity for incorporation of the L56-fragment through nucleophilic displacement of an appropriately derived electrophile, in this case benzyl mesylate 11. While initial transformations required some optimization, no additional modifications to the original route were necessary, effectively delivering 04SBT04 for biochemical evaluation.

[0144] An assay of the dissociation constant (K _D ) of 04SBT04 to GTP-bound RAS will ascertain potency relative to 31 MEW44 structure shown below:

Example 3

[0145] Evaluating Covalent Inhibition at L56 Site. While analyzing the L56 site in pursuit of a novel 3-site compound, the presence of several potentially nucleophilic residues lining this pocket was noted, namely K5. There was a possibility of developing irreversible inhibitors at L56 and assaying the pharmacological consequences of covalently modifying RAS at this site. This approach hinged upon the ability to introduce adequately positioned electrophihc moieties into a scaffold already possessing appreciable affinity for the targeted pocket. Since the L56-fragment had been separately validated by Sun et al. and our independent virtual screens, it was seen as an appropriate scaffold for evaluating the hypothesis. Given the observed docking pose of this fragment, it was reasoned that derivation of the indole ring with electrophihc moieties gave the greatest likelihood of effectively engaging a nucleophilic residue. These electrophile-derived fragments could be incorporated into analogs of 04SBT04, potentially generating highly selective covalent inhibitors.

[0146] The synthetic routes towards these electrophihc fragments are depicted in Scheme 2 (FIG. 7), relying on amino-substituted indole analog 18. After modifications of the synthetic route towards L56-fragment, the aryl amine analog with electrophihc warheads could effectively be derived to arrive at the target probes for assessing irreversible inhibition. Mass spectroscopy experiments capable of discerning covalent modification of RAS will be conducted.

[0147] In order to effectively capture the vast landscape of challenging, yet disease-modifying, therapeutic targets, innovative approaches to drug discovery are paramount. Work in this field suggests that rational, structure-guided design of multivalent ligands capable of mitigating protein-protein interactions is a viable option. The current strategy resulted in the generation of pan-RAS inhibitors demonstrating intriguing activity in biochemical, in vitro, and in vivo assays. Documents

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[0148] All patents, patent applications, and publications cited above are incorporated herein by reference in their entirety as if recited in full herein.

[0149] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention and all such modifications are intended to be included within the scope of the following claims.