Compounds, compositions, methods for treating diseases, and methods for preparing compounds

Some embodiments of the invention include inventive compounds (e.g., compounds of Formula (I)). Other embodiments include compositions (e.g., pharmaceutical compositions) comprising the inventive compound. Still other embodiments of the invention include compositions (e.g., pharmaceutical compositions) for treating, for example, certain diseases using the inventive compounds. Some embodiments include methods of using the inventive compound (e.g., in compositions or in pharmaceutical compositions) for administering and treating (e.g., diseases such as cancer). Further embodiments include methods for making the inventive compounds. Additional embodiments of the invention are also discussed herein.

BACKGROUND

Some Ras protein mutations can give rise to cancer. Over 90% of pancreatic tumors appear to be associated with some Ras protein mutations. In certain instances, when Ras protein is bound by GTP, Ras guanine nucleotide exchange factors (RasGEFs) appear to activate Ras-like (Ral) proteins by promoting Ral protein binding to GTP. Activated Ral proteins can sometimes enhance the development of cancer, including tumorigenesis and metastasis. There are four members of the RalGEF family: Ral guanine nucleotide dissociation stimulator (RalGDS), RGL1, RGL2, and RGL3.

Several compounds are known to treat cancer, but do so inadequately. For example, many attempts to develop a clinically effective Ras inhibitor have failed. There appear to be no reported inhibitors of RalGEFs.

Certain embodiments of the invention address one or more of the deficiencies described above. For example, in some embodiments of the invention, inventive compounds such as Formula (I) are disclosed. In some embodiments, RalGEF inhibitors are disclosed. Other embodiments include compositions (e.g., pharmaceutical compositions) comprising the inventive compound. Still other embodiments of the invention include compositions (e.g., pharmaceutical compositions) for treating, for example, certain diseases using the inventive compounds. Some embodiments include methods of using the inventive compound (e.g., in compositions or in pharmaceutical compositions) for administering and treating (e.g., diseases such as cancer). Further embodiments include methods for making the inventive compounds. Additional embodiments of the invention are also discussed herein.

SUMMARY

Some embodiments of the present invention include a compound selected from Formula (I):

In yet other embodiments, X is

In some embodiments, ring A is

In other embodiments, ring A is

or ring A is not

In certain embodiments, compound I-1 is excluded from Formula (I).

Some embodiments of the invention include a composition comprising a compound, as disclosed herein (e.g., Formula (I)). In certain embodiments, the amount of the compound is from about 0.0001% (by weight total composition) to about 99%. In other embodiments, the composition further comprises a formulary ingredient, an adjuvant, or a carrier.

Some embodiments of the invention include a pharmaceutical composition comprising a compound, as disclosed herein (e.g., Formula (I)). In some embodiments, the amount of the compound is from about 0.0001% (by weight total composition) to about 50%. In other embodiments, the pharmaceutical composition further comprises a formulary ingredient, an adjuvant, or a carrier.

Some embodiments of the invention include a method for providing an animal with a compound comprising one or more administrations of one or more compositions comprising a compound as disclosed herein (e.g., Formula (I)), where the compositions may be the same or different if there is more than one administration. In other embodiments, at least one of the one or more compositions further comprises a formulary ingredient. In certain embodiments, at least one of the one or more compositions comprises a composition as disclosed herein or a pharmaceutical composition as disclosed herein. In still other embodiments, at least one of the one or more administrations comprises parenteral administration, a mucosal administration, intravenous administration, subcutaneous administration, topical administration, intradermal administration, oral administration, sublingual administration, intranasal administration, or intramuscular administration. In other embodiments, if there is more than one administration at least one composition used for at least one administration is different from the composition of at least one other administration. In certain embodiments, the compound of at least one of the one or more compositions is administered to the animal in an amount of from about 0.01 mg/kg animal body weight to about 15 mg/kg animal body weight. In yet still other embodiments, the animal is a human, a rodent, or a primate.

Some embodiments of the invention include a method for treating an animal for a disease, comprising one or more administrations of one or more compositions comprising a compound as disclosed here (e.g., Formula (I)), where the compositions may be the same or different if there is more than one administration. In some embodiments, at least one of the one or more compositions further comprises a formulary ingredient. In other embodiments, at least one of the one or more compositions comprises a composition as disclosed herein or a pharmaceutical composition as disclosed herein. In yet other embodiments, at least one of the one or more administrations comprises parenteral administration, a mucosal administration, intravenous administration, subcutaneous administration, topical administration, intradermal administration, oral administration, sublingual administration, intranasal administration, or intramuscular administration. In still other embodiments, if there is more than one administration at least one composition used for at least one administration is different from the composition of at least one other administration. In other embodiments, the compound of at least one of the one or more compositions is administered to the animal in an amount of from about 0.005 mg/kg animal body weight to about 50 mg/kg animal body weight. In some embodiments, the animal is a human, a rodent, or a primate. In certain embodiments, the animal is in need of the treatment. In other embodiments, the method is for treating cancer. In yet other embodiments, the method is for treating pancreatic cancer, pancreatic ductal adenocarcinoma, lung cancer, liver cancer, colorectal cancer, colon cancer, rectal cancer, melanoma, cutaneous malignant melanoma, melanoma tumorigenesis, bladder cancer, prostate cancer, malignant nerve sheath tumors, multiple myeloma, breast cancer, squamous cell carcinoma, head and neck squamous cell carcinoma, lymphoma, leukemia, bone marrow cancer, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, glioblastoma multiforme, endometrial cancer, kidney cancer, basal cell carcinoma, thyroid cancer, neuroblastoma, ovarian cancer, renal cell carcinoma, hepatocellular carcinoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia, rhabdomyosarcoma, meningioma, gastric cancer, Glioma, oral cancer, nasopharyngeal carcinoma, stomach cancer, uterine cancer, medulloblastoma, cancers that can result in metastasis, cancers resulting from metastasis, or cancerous tumors thereof. In yet other embodiments, the method is for treating pancreatic cancer, pancreatic ductal adenocarcinoma, lung cancer, liver cancer, colorectal cancer, colon cancer, rectal cancer, melanoma, cutaneous malignant melanoma, melanoma tumorigenesis, bladder cancer, malignant nerve sheath tumors, multiple myeloma, breast cancer, squamous cell carcinoma, head and neck squamous cell carcinoma, ovarian cancer, prostate cancer, medulloblastoma, cancers that can result in metastasis, cancers resulting from metastasis, or cancerous tumors thereof. In certain embodiments, the method is for treating pancreatic cancer, pancreatic ductal adenocarcinoma, lung cancer, liver cancer, ovarian cancer, prostate cancer, medulloblastoma, cancers that can result in metastasis, cancer resulting from metastasis of pancreatic cancer, lung cancer resulting from metastasis, or cancerous tumors thereof.

Some embodiments of the invention include a method for preparing a compound as disclosed herein (e.g., Formula (I)) comprising,(a) reacting a compound of Formula (II) with a compound of Formula (III) to result in a mixture comprising a compound of Formula (IV);(b) reacting a compound of Formula (IV) with a suitable compound to convert an oxo to a halogen to result in a mixture comprising a compound of Formula (V);(c) reacting a compound of Formula (V) with a compound of Formula (VI); and;(d) recovering Formula (I),
where Formula (II) is

and R11is a halogen or —CH2-halogen;

Formula (IV) is

Formula (V) is

Formula (VI) is

In some embodiments, R11is —CH2-halogen, —CH2Br, or —CH2Cl. In other embodiments, the suitable compound to convert an oxo to a halogen is POCl3or POBr3.

Other embodiments of the invention are also discussed herein.

DETAILED DESCRIPTION

While embodiments encompassing the general inventive concepts may take diverse forms, various embodiments will be described herein, with the understanding that the present disclosure is to be considered merely exemplary, and the general inventive concepts are not intended to be limited to the disclosed embodiments.

Some embodiments of the invention include inventive compounds (e.g., compounds of Formula (I)). Other embodiments include compositions (e.g., pharmaceutical compositions) comprising the inventive compound. Still other embodiments of the invention include compositions for treating, for example, certain diseases using the inventive compounds. Some embodiments include methods of using the inventive compound (e.g., in compositions or in pharmaceutical compositions) for administering and treating. Further embodiments include methods for making the inventive compound.

As used herein (unless otherwise specified), the term “alkoxy” means any of the above alkyl groups which is attached to the remainder of the molecule by an oxygen atom (alkyl-O—). Examples of alkoxy groups include, but are not limited to, methoxy (sometimes shown as MeO—), ethoxy, isopropoxy, propoxy, and butyloxy.

As used herein (unless otherwise specified), the term “alkynyl” means a monovalent, straight or branched hydrocarbon chain that includes one or more (e.g., 1, 2, 3, or 4) triple bonds and that also may optionally include one or more (e.g. 1, 2, 3, or 4) double bonds in the chain. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, and 5-hexynyl.

As used herein (unless otherwise specified), the term “aryl” means a monovalent, monocyclic or bicyclic, 5, 6, 7, 8, 9, 10, 11, or 12 member aromatic hydrocarbon group which, when unsubstituted. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tolyl, and xylyl. For an bicyclic aryl that is designated as substituted, one or both rings can be substituted.

As used herein (unless otherwise specified), the term “cycloalkyl” means a monovalent, monocyclic or bicyclic, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 membered hydrocarbon group. The rings can be saturated or partially unsaturated. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and bicycloalkyls (e.g., bicyclooctanes such as [2.2.2]bicyclooctane or [3.3.0]bicyclooctane, bicyclononanes such as [4.3.0]bicyclononane, and bicyclodecanes such as [4.4.0]bicyclodecane (decalin), or spiro compounds). For a monocyclic cycloalkyl, the ring is not aromatic. For a bicyclic cycloalkyl, if one ring is aromatic, then the other is not aromatic. For a bicyclic cycloalkyl that is designated as substituted, one or both rings can be substituted.

As used herein (unless otherwise specified), the term “halogen” means monovalent Cl, F, Br, or I.

As used herein (unless otherwise specified), the term “heteroaryl” means a monovalent, monocyclic or bicyclic, 5, 6, 7, 8, 9, 10, 11, or 12 membered, hydrocarbon group, where 1, 2, 3, 4, 5, or 6 carbon atoms are replaced by a hetero atom independently selected from nitrogen, oxygen, or sulfur atom, and the monocyclic or bicyclic ring system is aromatic. Examples of heteroaryl groups include, but are not limited to, thienyl (or thiophenyl), furyl, indolyl, pyrrolyl, pyridinyl, pyrazinyl, oxazolyl, thiaxolyl, quinolinyl, pyrimidinyl, imidazolyl, triazolyl, tetrazolyl, 1H-pyrazol-4-yl, 1-Me-pyrazol-4-yl, pyridin-3-yl, pyridin-4-yl, 3,5-dimethylisoxazolyl, 1H-pyrrol-3-yl, 3,5-di-Me-pyrazolyl, and 1H-pyrazol-4-yl. For a bicyclic heteroaryl, if one ring is aryl, then the other is heteroaryl. For a bicyclic heteroaryl, one or both rings can have one or more hetero atoms. For a bicyclic heteroaryl that is designated as substituted, one or both rings can be substituted.

As used herein (unless otherwise specified), the term “heterocyclyl” means a monovalent, monocyclic or bicyclic, 5, 6, 7, 8, 9, 10, 11, or 12 membered, hydrocarbon, where 1, 2, 3, 4, 5, or 6 carbon atoms are replaced by a hetero atom independently selected from nitrogen atom, oxygen atom, or sulfur atom, and the monocyclic or bicyclic ring system is not aromatic. Examples of heterocyclyl groups include, but are not limited to, tetrahydropyran, pyrolidinyl (e.g., pyrrolidin-1-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, or pyrrolidin-4-yl), piperazinyl (e.g., piperazin-1-yl, piperazin-2-yl, piperazin-3-yl, or piperazin-4-yl), piperidinyl (e.g., piperadin-1-yl, piperadin-2-yl, piperadin-3-yl, or piperadin-4-yl), and morpholinyl (e.g., morpholin-1-yl, morpholin-2-yl, morpholin-3-yl, or morpholin-4-yl). For a bicyclic heterocyclyl, if one ring is aromatic (e.g., monocyclic aryl or heteroaryl), then the other ring is not aromatic. For a bicyclic heterocyclyl, one or both rings can have one or more hetero atoms. For a bicyclic heterocyclyl that is designated as substituted, one or both rings can be substituted.

As used herein (unless otherwise specified), the term “hetero atom” means an atom selected from nitrogen atom, oxygen atom, or sulfur atom.

As used herein (unless otherwise specified), the terms “hydroxy” or “hydroxyl” indicates the presence of a monovalent —OH group.

As used herein (unless otherwise specified), the term “substituted” (e.g., as in substituted alkyl) means that one or more hydrogen atoms of a chemical group (with one or more hydrogen atoms) can be replaced by one or more non-hydrogen substituents selected from the specified options. The replacement can occur at one or more positions. The term “optionally substituted” means that one or more hydrogen atoms of a chemical group (with one or more hydrogen atoms) can be, but is not required to be substituted.

Some compounds of the invention can have one or more chiral centers and can exist in and be isolated in optically active and racemic forms, for any of the one or more chiral centers. Some compounds can exhibit polymorphism. The compounds of the present invention (e.g., Formula I) encompass any optically active, racemate, stereoisomer form, polymorphism, or mixtures thereof. If a chiral center does not provide an indication of its configuration (i.e., R or S) in a chemical structure, it should be considered to represent R, S or a racemate.

Compounds and Compositions Including Pharmaceutical Compositions

Some embodiments of the invention include compounds of Formula (I):

In some embodiments, X can be

In certain embodiments, X can be

Any of the above X moieties can be inserted into the structure as shown or can be rotated 180 degrees along a vertical axis and then inserted into the structure (e.g.,

can be rotated 180 degrees along a vertical axis to

and then inserted into the structure).

In some embodiments, ring A is a monocyclic or bicyclic ring comprising 4, 5, 6, 7, 8, 9, or 10 carbon atoms (e.g., 4, 5, 6, 7, or 8 carbon atoms), where 1, 2, 3, 4, or 5 (e.g., 1, 2, or 3) of the non-fusion carbon atoms, can be optionally replaced with a hetero atom (e.g., N, O, S) and the hetero atom can be the same or different if more than one carbon atom is replaced, which ring A carbon atoms or any of the hetero atom replacements (as chemically appropriate) can optionally be substituted with one or more (e.g., 0, 1, 2, 3, 4, 5, or 6) of halogen (e.g., F, Cl, Br, or I), oxo (═O), hydroxy (—OH), methanoyl (—COH), carboxy (—CO2H), nitro (—NO2), —NH2, —N(CH3)2, cyano (—CN), ethynyl (—CCH), propynyl, sulfo (—SO3H), morpholinyl, —CO-morpholin-4-yl, —CONH2, —CON(CH3)2, C1-C6alkyl, C1-C6perfluoronated alkyl (e.g., trifluoromethyl or perfluoroethyl), or C1-C5alkoxy. The two “fusion carbon atoms” are defined as the two ring A carbon atoms which fuse ring A to the pyrimidine-like moiety; “non-fusion carbon atoms” are defined as the other carbon atoms in ring A that are not the two fusion carbon atoms. Ring A can be monocyclic or bicyclic, to create (i.e., together with the pyrimidine-like moiety) a bicyclic or tricyclic structure, respectively. Ring A can, in certain embodiments, comprise 1, 2, 3, 4, or 5 double bonds, which can be in one or both rings if Ring A is bicyclic. In other embodiments, ring A can itself be aromatic, can create an aromatic of the bi- or tri-cyclic (i.e., ring A fused with the pyrimidine-like moiety), or both. In certain embodiments, the fusion carbon atoms can be single bonded or double bonded to each other. In some embodiments, ring A can be

In some embodiments, ring A can be

As used herein, * is defined to indicate a point of fusion for each of the two fusion carbon atoms of ring A. Any of the above moieties can be inserted into the structure as shown or can be rotated 180 degrees along a horizontal axis and then inserted into the structure (e.g.,

can be rotated 180 degrees along a horizontal axis to

and then inserted into the structure).

In some embodiments, the compounds of Formula (I) can be selected from those specified in Table 1.

In some embodiments, R1is not H, R2is not H, R3is not H, R4is not 3-methoxyphenyl, R5is not 3-methoxyphenyl, X is not

or ring A is not

In other embodiments, one or more (e.g., one, two, three, or four) of the following provisos apply to Formula (I): R1is not H, R2is not H, R3is not H, R4is not 3-methoxyphenyl, R5is not 3-methoxyphenyl, X is not

or ring A is not

In some embodiments, R5is not 3-methoxyphenyl. In other embodiments, X is not

In some embodiments, the compounds of Formula (I) can be in the form of salts, optical and geometric isomers, and salts of isomers. In other embodiments, the compounds can be in various forms, such as uncharged molecules, components of molecular complexes, or non-irritating pharmacologically acceptable salts, including but not limited to hydrochloride, hydrobromide, sulphate, phosphate, nitrate, borate, acetate, maleate, tartrate, and salicylate. In some instances, for acidic compounds, salts can include metals, amines, or organic cations (e.g. quaternary ammonium). In yet other embodiments, simple derivatives of the compounds (e.g., ethers, esters, or amides) which have desirable retention and release characteristics but which are easily hydrolyzed by body pH, enzymes, or other suitable means, can be employed.

In some embodiments, the compounds of the invention having a chiral center and can exist in and be isolated in optically active and racemic forms. In other embodiments, compounds may exhibit polymorphism. Some embodiments of the present invention encompass any racemic, optically active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound described herein. The preparation of optically active forms can be accomplished by any suitable method, including but not limited to, resolution of the racemic form by recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase.

In some embodiments, the compounds of the invention (e.g., Formula (I), I-1, or I-2) can inhibit the activity of a member of the RalGEF family (e.g., RalGDS, RGL1, RGL2, RGL3) or a combination thereof. In certain embodiments, the compounds of the invention (e.g., Formula (I), I-1, or I-2) can inhibit the activity of RalGDS and RGL2. In other embodiments, the compounds of the invention (e.g., Formula (I), I-1, or I-2) can be a pan-RalGEF inhibitor. In other embodiments, the compounds of the invention (e.g., Formula (I), I-1, or I-2) can inhibit the interaction of K-Ras with RalGDS and can inhibit the interaction of K-Ras with RGL2. In still other embodiments, the compounds of the invention (e.g., Formula (I), I-1, or I-2) can promote cell cycle arrest/senescence, promote apoptosis, suppress Ral induced secretion, suppress the activation of the NFkB pathway in Ras driven pancreatic carcinoma cells, block the growth and development of cancer stem cells, suppress mitochondrial fission components, or a combination thereof.

In certain embodiments, one or more compounds of the invention (e.g., Formula (I)) can be part of a composition and can be in an amount (by weight of the total composition) of at least about 0.0001%, at least about 0.001%, at least about 0.10%, at least about 0.15%, at least about 0.20%, at least about 0.25%, at least about 0.50%, at least about 0.75%, at least about 1%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, at least about 99%, at least about 99.99%, no more than about 75%, no more than about 90%, no more than about 95%, no more than about 99%, or no more than about 99.99%, from about 0.0001% to about 99%, from about 0.0001% to about 50%, from about 0.01% to about 95%, from about 1% to about 95%, from about 10% to about 90%, or from about 25% to about 75%.

In some embodiments, one or more compounds of the invention (e.g., Formula (I)) can be purified or isolated in an amount (by weight of the total composition) of at least about 0.0001%, at least about 0.001%, at least about 0.10%, at least about 0.15%, at least about 0.20%, at least about 0.25%, at least about 0.50%, at least about 0.75%, at least about 1%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, at least about 99%, at least about 99.99%, no more than about 75%, no more than about 90%, no more than about 95%, no more than about 99%, no more than about 99.99%, from about 0.0001% to about 99%, from about 0.0001% to about 50%, from about 0.01% to about 95%, from about 1% to about 95%, from about 10% to about 90%, or from about 25% to about 75%.

Some embodiments of the present invention include compositions comprising one or more compounds of the invention (e.g., Formula (I)). In certain embodiments, the composition is a pharmaceutical composition, such as compositions that are suitable for administration to animals (e.g., mammals, primates, monkeys, humans, canine, feline, porcine, mice, rabbits, or rats). In some instances, the pharmaceutical composition is non-toxic, does not cause side effects, or both. In some embodiments, there may be inherent side effects (e.g., it may harm the patient or may be toxic or harmful to some degree in some patients).

“Therapeutically effective amount” means an amount effective to achieve a desired and/or beneficial effect. An effective amount can be administered in one or more administrations. For some purposes of this invention, a therapeutically effective amount is an amount appropriate to treat an indication. By treating an indication is meant achieving any desirable effect, such as one or more of palliate, ameliorate, stabilize, reverse, slow, or delay disease progression, increase the quality of life, or to prolong life. Such achievement can be measured by any suitable method, such as measurement of tumor size.

In some embodiments, one or more compounds of the invention (e.g., Formula (I)) can be part of a pharmaceutical composition and can be in an amount of at least about 0.0001%, at least about 0.001%, at least about 0.10%, at least about 0.15%, at least about 0.20%, at least about 0.25%, at least about 0.50%, at least about 0.75%, at least about 1%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, at least about 99%, at least about 99.99%, no more than about 75%, no more than about 90%, no more than about 95%, no more than about 99%, no more than about 99.99%, from about 0.001% to about 99%, from about 0.001% to about 50%, from about 0.1% to about 99%, from about 1% to about 95%, from about 10% to about 90%, or from about 25% to about 75%. In some embodiments, the pharmaceutical composition can be presented in a dosage form which is suitable for the topical, subcutaneous, intrathecal, intraperitoneal, oral, parenteral, rectal, cutaneous, nasal, vaginal, or ocular administration route. In other embodiments, the pharmaceutical composition can be presented in a dosage form which is suitable for parenteral administration, a mucosal administration, intravenous administration, subcutaneous administration, topical administration, intradermal administration, oral administration, sublingual administration, intranasal administration, or intramuscular administration. The pharmaceutical composition can be in the form of, for example, tablets, capsules, pills, powders granulates, suspensions, emulsions, solutions, gels (including hydrogels), pastes, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables, implants, sprays, aerosols or other suitable forms.

In some embodiments, the pharmaceutical composition can include one or more formulary ingredients. A “formulary ingredient” can be any suitable ingredient (e.g., suitable for the drug(s), for the dosage of the drug(s), for the timing of release of the drugs(s), for the disease, for the disease state, or for the delivery route) including, but not limited to, water (e.g., boiled water, distilled water, filtered water, pyrogen-free water, or water with chloroform), sugar (e.g., sucrose, glucose, mannitol, sorbitol, xylitol, or syrups made therefrom), ethanol, glycerol, glycols (e.g., propylene glycol), acetone, ethers, DMSO, surfactants (e.g., anionic surfactants, cationic surfactants, zwitterionic surfactants, or nonionic surfactants (e.g., polysorbates)), oils (e.g., animal oils, plant oils (e.g., coconut oil or arachis oil), or mineral oils), oil derivatives (e.g., ethyl oleate, glyceryl monostearate, or hydrogenated glycerides), excipients, preservatives (e.g., cysteine, methionine, antioxidants (e.g., vitamins (e.g., A, E, or C), selenium, retinyl palmitate, sodium citrate, citric acid, chloroform, or parabens, (e.g., methyl paraben or propyl paraben)), or combinations thereof.

In certain embodiments, pharmaceutical compositions can be formulated to release the active ingredient (e.g., one or more compounds of the invention such as Formula (I)) substantially immediately upon the administration or any substantially predetermined time or time after administration. Such formulations can include, for example, controlled release formulations such as various controlled release compositions and coatings.

Other formulations (e.g., formulations of a pharmaceutical composition) can, in certain embodiments, include those incorporating the drug (or control release formulation) into food, food stuffs, feed, or drink.

Other embodiments of the invention can include methods of administering or treating an organism, which can involve treatment with an amount of at least one compound of the invention (e.g., Formula (I)) that is effective to treat the disease, condition, or disorder that the organism has, or is suspected of having, or is susceptible to, or to bring about a desired physiological effect. In some embodiments, the composition or pharmaceutical composition comprises at least one compound of the invention (e.g., Formula (I)) which can be administered to an animal (e.g., mammals, primates, monkeys, or humans) in an amount of about 0.005 to about 50 mg/kg body weight, about 0.01 to about 15 mg/kg body weight, about 0.1 to about 10 mg/kg body weight, about 0.5 to about 7 mg/kg body weight, about 0.005 mg/kg, about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 3 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 10 mg/kg, about 12 mg/kg, or about 15 mg/kg. In regard to some conditions, the dosage can be about 0.5 mg/kg human body weight or about 6.5 mg/kg human body weight. In some instances, some animals (e.g., mammals, mice, rabbits, feline, porcine, or canine) can be administered a dosage of about 0.005 to about 50 mg/kg body weight, about 0.01 to about 15 mg/kg body weight, about 0.1 to about 10 mg/kg body weight, about 0.5 to about 7 mg/kg body weight, about 0.005 mg/kg, about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 80 mg/kg, about 100 mg/kg, or about 150 mg/kg. Of course, those skilled in the art will appreciate that it is possible to employ many concentrations in the methods of the present invention, and using, in part, the guidance provided herein, will be able to adjust and test any number of concentrations in order to find one that achieves the desired result in a given circumstance. In other embodiments, the compounds of the invention (e.g., Formula (I)) can be administered in combination with one or more other therapeutic agents for a given disease, condition, or disorder.

In some embodiments, the compositions can include a unit dose of one or more compounds of the invention (e.g., Formula (I)) in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and excipients. In certain embodiments, the carrier, vehicle or excipient can facilitate administration, delivery and/or improve preservation of the composition. In other embodiments, the one or more carriers, include but are not limited to, saline solutions such as normal saline, Ringer's solution, PBS (phosphate-buffered saline), and generally mixtures of various salts including potassium and phosphate salts with or without sugar additives such as glucose. Carriers can include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. In other embodiments, the one or more excipients can include, but are not limited to water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Nontoxic auxiliary substances, such as wetting agents, buffers, or emulsifiers may also be added to the composition. Oral formulations can include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate.

Administration Routes and Treatments of Disease

The compounds of the invention (e.g., Formula (I), I-1, or I-2) can be administered to animals by any number of suitable administration routes or formulations. The compounds of the invention (e.g., Formula (I), I-1, or I-2) can also be used to treat animals for a variety of diseases. Animals include but are not limited to mammals, primates, monkeys (e.g., macaque, rhesus macaque, or pig tail macaque), humans, canine, feline, bovine, porcine, avian (e.g., chicken), mice, rabbits, and rats. As used herein, the term “subject” refers to both human and animal subjects.

The route of administration of the compounds of the invention (e.g., Formula (I), I-1, or I-2) can be of any suitable route. Administration routes can be, but are not limited to the oral route, the parenteral route, the cutaneous route, the nasal route, the rectal route, the vaginal route, and the ocular route. In other embodiments, administration routes can be parenteral administration, a mucosal administration, intravenous administration, subcutaneous administration, topical administration, intradermal administration, oral administration, sublingual administration, intranasal administration, or intramuscular administration. The choice of administration route can depend on the compound identity (e.g., the physical and chemical properties of the compound) as well as the age and weight of the animal, the particular disease (e.g., cancer), and the severity of the disease (e.g., stage or severity of cancer). Of course, combinations of administration routes can be administered, as desired.

Some embodiments of the invention include a method for providing a subject with a composition comprising one or more compounds of the invention (e.g., Formula (I), I-1, or I-2) described herein (e.g., a pharmaceutical composition) which comprises one or more administrations of one or more such compositions; the compositions may be the same or different if there is more than one administration.

Diseases that can be treated in an animal (e.g., mammals, porcine, canine, avian (e.g., chicken), bovine, feline, primates, rodents, monkeys, rabbits, mice, rats, and humans) using a compound of the invention (e.g., Formula (I), I-1, or 1-2) include, but are not limited to cancers.

In some embodiments, cancers that can be treated in an animal (e.g., mammals, porcine, canine, avian (e.g., chicken), bovine, feline, primates, rodents, monkeys, rabbits, mice, rats, and humans) using a compound of the invention (e.g., Formula (I), I-1, or I-2) include, but are not limited to cancers associated with a RAS mutation. In some embodiments, cancers that can be treated include, but are not limited to, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, prostate cancer, malignant nerve sheath tumors, multiple myeloma, breast cancer, squamous cell carcinoma (e.g., head and neck squamous cell carcinoma), lymphoma, leukemia, bone marrow cancer, non-Hodgkin lymphoma (e.g., diffuse large B-cell lymphoma), glioblastoma multiforme, endometrial cancer, kidney cancer, basal cell carcinoma, thyroid cancer, neuroblastoma, ovarian cancer, renal cell carcinoma, hepatocellular carcinoma, colon cancer, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia, rhabdomyosarcoma, meningioma, gastric cancer, Glioma, oral cancer, nasopharyngeal carcinoma, rectal cancer, stomach cancer, uterine cancer, medulloblastoma, cancers that can result in metastasis, cancers resulting from metastasis, or cancerous tumors thereof. In some embodiments, cancers that can be treated include, but are not limited to, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, malignant nerve sheath tumors, multiple myeloma, breast cancer, squamous cell carcinoma (e.g., head and neck squamous cell carcinoma), ovarian cancer, prostate cancer, medulloblastoma, cancers that can result in metastasis (e.g., pancreatic cancer), cancers resulting from metastasis (e.g., lung cancer), or cancerous tumors thereof. In some embodiments, cancers that can be treated include, but are not limited to, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof. In some embodiments, cancers that can be treated include, but are not limited to, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma) or cancerous tumors thereof. Animals that can be treated include but are not limited to mammals, rodents, primates, monkeys (e.g., macaque, rhesus macaque, pig tail macaque), humans, canine, feline, porcine, avian (e.g., chicken), bovine, mice, rabbits, and rats. As used herein, the term “subject” refers to both human and animal subjects. In some instances, the animal is in need of the treatment (e.g., by showing signs of disease or cancer, or by having a cancerous tumor).

In some embodiments, cancers that can be treated in an animal (e.g., mammals, porcine, canine, avian (e.g., chicken), bovine, feline, primates, rodents, monkeys, rabbits, mice, rats, and humans) using a compound of the invention (e.g., Formula (I), I-1, or I-2) include, but are not limited to cancers that can be treated by inhibiting (e.g., reducing the activity or expression of) a member of the RalGEF family (e.g., RalGDS or RGL2), by inhibiting RalGDS, by inhibiting RGL2, or combinations thereof. In some embodiments, cancers that can be treated in an animal include cancers that can be treated by inhibiting both RalGDS and RGL2.

As used herein, the term “treating” (and its variations, such as “treatment”) is to be considered in its broadest context. In particular, the term “treating” does not necessarily imply that an animal is treated until total recovery. Accordingly, “treating” includes amelioration of the symptoms, relief from the symptoms or effects associated with a condition, decrease in severity of a condition, or preventing, preventively ameliorating symptoms, or otherwise reducing the risk of developing a particular condition. As used herein, reference to “treating” an animal includes but is not limited to prophylactic treatment and therapeutic treatment. Any of the compositions (e.g., pharmaceutical compositions) described herein can be used to treat an animal.

As related to treating cancer (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof), treating can include but is not limited to prophylactic treatment and therapeutic treatment. As such, treatment can include, but is not limited to: preventing cancer (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof); reducing the risk of cancer (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof); ameliorating or relieving symptoms of cancer (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof); eliciting a bodily response against cancer (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof); inhibiting the development or progression of cancer (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof); inhibiting or preventing the onset of symptoms associated with cancer (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof); reducing the severity of cancer (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof); causing a regression of cancer (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof) or one or more of the symptoms associated with cancer (e.g., a decrease in tumor size); causing remission of cancer (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof); or preventing relapse of cancer (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof). In some embodiments, treating does not include prophylactic treatment of cancer (e.g., preventing or ameliorating future cancer).

Treatment of an animal can occur using any suitable administration method (such as those disclosed herein) and using any suitable amount of a compound of the invention (e.g., Formula (I), I-1, or I-2). In some embodiments, methods of treatment comprise treating an animal for cancer (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, liver cancer, colorectal cancer (e.g., colon cancer or rectal cancer), melanoma (e.g., cutaneous malignant melanoma, melanoma tumorigenesis), bladder cancer, ovarian cancer, prostate cancer, medulloblastoma, or cancerous tumors thereof). Some embodiments of the invention include a method for treating a subject (e.g., an animal such as a human or primate) with a composition comprising a compound of the invention (e.g., Formula (I), I-1, or I-2) (e.g., a pharmaceutical composition) which comprises one or more administrations of one or more such compositions; the compositions may be the same or different if there is more than one administration.

“Therapeutically effective amount” means an amount effective to achieve a desired and/or beneficial effect (e.g., decreasing tumor size). A therapeutically effective amount can be administered in one or more administrations. For some purposes of this invention, a therapeutically effective amount is an amount appropriate to treat an indication (e.g., to treat cancer). By treating an indication is meant achieving any desirable effect, such as one or more of palliate, ameliorate, stabilize, reverse, slow, or delay disease (e.g., cancer) progression, increase the quality of life, or to prolong life. Such achievement can be measured by any suitable method, such as but not limited to measurement of tumor size.

In some embodiments, the treatments can also include one or more of surgical intervention, chemotherapy, radiation therapy, hormone therapies, immunotherapy, and adjuvant systematic therapies. Adjuvants may include but are not limited to chemotherapy (e.g., temozolomide), radiation therapy, antiangiogenic therapy (e.g., bevacizumab), and hormone therapies, such as administration of LHRH agonists; antiestrogens, such as tamoxifen; high-dose progestogens; aromatase inhibitors; and/or adrenalectomy. Chemotherapy can be used as a single-agent or as a combination with known or new therapies.

In some embodiments, the administration of at least one compound of the invention (e.g., Formula (I), I-1, or I-2) is an adjuvant cancer therapy or part of an adjuvant cancer therapy. Adjuvant treatments include treatments by the mechanisms disclosed herein and of cancers as disclosed herein, including, but not limited to tumors. Corresponding primary therapies can include, but are not limited to, surgery, chemotherapy, or radiation therapy. In some instances, the adjuvant treatment can be a combination of chemokine receptor antagonists with traditional chemotoxic agents or with immunotherapy that increases the specificity of treatment to the cancer and potentially limits additional systemic side effects. In still other embodiments, a compound of the invention (e.g., Formula (I), I-1, or I-2) can be used as adjuvant with other chemotherapeutic agents. The use of a compound of the invention (e.g., Formula (I), I-1, or I-2) may, in some instances, reduce the duration of the dose of both drugs and drug combinations reducing the side effects.

In some embodiments, the treatments disclosed herein can include use of other drugs (e.g., antibiotics) or therapies for treating disease. For example, antibiotics can be used to treat infections and can be combined with a compound of the invention to treat disease (e.g., infections associated with cancer). In other embodiments, intravenous immunoglobulin (IVIG) therapy can be used as part of the treatment regime (i.e., in addition to administration of the compound(s) of the invention).

Methods for Preparing Compounds of Formula (I)

Some embodiments of the present invention include methods for the preparation of compounds of Formula (I). The compounds of Formula (I) can be prepared using any suitable method or they can be purchased, if available. In certain embodiments, a compound of Formula (I) can be prepared comprising the step of reacting a compound of Formula (II) with a compound of Formula (III) to result in Formula (IV), which is later made into Formula (I) (e.g., using one or more synthetic steps).

In some embodiments R11can be halogen (e.g., F, Cl, Br, or I) or —CH2-halogen. In other embodiments, R11can be Cl or —CH2Cl. In still other embodiments, R11can be —CH2-halogen, —CH2Br, or —CH2Cl. R5, X, and ring A of Formulas (II), (III), and (IV) are the same as that defined in Formula (I). Formula (II) can be prepared using any suitable method or can be purchased if available. Formula (III) can be prepared using any suitable method or can be purchased where available.

In some embodiments, Formula (II) can be reacted with Formula (III) under the following conditions: Formula (II) and Formula (III) can be in a mixture comprising a solvent (e.g., DMF). The mixture can be heated (e.g., using a microwave) at a certain temperature (e.g., about 120° C.) for a certain amount of time (e.g., about 2 hours). Formula (IV) can then optionally be recovered.

In some embodiments, Formula (III) (e.g., about 1.3 mL or about 1.34 g or about 8.86 mmol) is added to Formula (II) (e.g., about 718 mg or about 3.69 mmol) in about 6 mL (e.g., from about 1 mL to about 20 mL) of DMF (or any suitable solvent). The resulting solution is stirred at about 120° C. (or from about 90° C. to about 150° C.) for about 2 hours (or from about 0.5 hours to about 10 hours) under heat (e.g., microwave irradiation). After cooling to room temperature, the mixture can be poured into about 100 mL (or from about 20 mL to about 500 ml) of water. The aqueous mixture can stand for about 5 minutes (or from about 1 minute to about 25 minutes) and then be extracted three times (or from one time to 10 times) with about 75 mL portions (or from about 10 mL to about 500 mL) of ethyl acetate (or any suitable solvent). The combined organic layers can then be washed (e.g., with brine), dried (e.g., with Na2SO4) and concentrated (e.g., in vacuo). The crude product can be dissolved in minimal amount of ethyl acetate (or any suitable solvent) and can then be precipitated (e.g., by adding drops of hexanes (or any suitable solvent)) to provide Formula (IV).

R5, X, and ring A of Formula (IV) and Formula (V) are the same as that defined in Formula (I). Formula (IV) can be prepared using any suitable method (e.g., see above) or can be purchased if available.

In some embodiments, Formula (IV) can be reacted to provide Formula (V) under the following conditions: Formula (IV) can be mixed with POCl3(or any suitable compound to convert the oxo of Formula (IV) to Cl or other halogen) and is heated (e.g., with a microwave) at a certain temperature (e.g., about 90° C.) for a certain amount of time (e.g., about 3 hours). Formula (V) can then optionally be recovered.

Formula (IV) (e.g., about 309 mg or about 0.971 mmol) in about 3 mL (or from about 0.5 mL to about 20 mL) of POCl3(or any suitable compound to convert the oxo of Formula (IV) to Cl or other halogen, such as POBr3) can be stirred at about 90° C. (or from about 30° C. to about 150° C.) for about 3 hours (or from about 0.5 hours to about 20 hours) under heat (e.g., microwave irradiation). After cooling (e.g., to room temperature), the solvent can be removed (e.g., under reduced pressure). The residue can be diluted with about 10 mL (or from about 2 mL to about 50 mL) of water (e.g., cold water) containing about 2 g (or from about 0.5 g to about 10 g) of a suitable salt (e.g., potassium carbonate). After sitting for about 5 minutes (or from about 0.5 minutes to about 50 minutes), the aqueous solution can be extracted twice (or once or up to 10 times) with about 10 mL (or from about 1 mL to about 100 mL) portions of any suitable solvent (e.g., ethyl acetate). The combined organic layers can be dried (e.g., using Na2SO4) and can be concentrated (e.g., in vacuo). The concentrate can then be chromatographed (e.g., over silica gel (Combiflash, 4 g column, 0-70%, hexanes-ethyl acetate, 10 minutes)) to provide Formula (V).

R1, R2, R3, R4, R5, X and ring A of Formula (V) and Formula (VI) are the same as that defined in Formula (I). Formula (V) can be prepared using any suitable method (e.g., see above) or can be purchased where available. Formula (VI) can be prepared using any suitable method or can be purchased if available.

In some embodiments, Formula (V) can be reacted with Formula (VI) to provide Formula (I) under the following conditions: Formula (V) and Formula (VI) can be in a mixture and heated (e.g., using a microwave) at a certain temperature (e.g., about 100° C.) for a certain amount of time (e.g., about 15 hours). Formula (I) can then optionally be recovered.

Formula (V) (e.g., about 208 mg or about 0.636 mmol) in about 20 mL (or from about 1 mL to about 200 mL) of tetrahydrofuran (or any suitable solvent) can be added to Formula (VI) (e.g., about 0.11 mL or about 113 mg or about 0.827 mmol) and to trimethylamine (or any suitable solvent) (e.g., about 0.44 mL or about 321 mg or about 3.18 mmol). The resulting solution can be stirred at about 100° C. (or from about 40° C. to about 300° C.) for about 15 hours (or about 2 hours to about 100 hours) under heat (e.g., microwave irradiation) and then can be cooled (e.g., to room temperature). The mixture can then be diluted with about 20 mL (or from about 2 mL to about 200 mL) of any suitable solvent (e.g., ethyl acetate) and then can be washed with water (e.g., washed 1, 2, 3, 4, or 5 times). The organic phase can be dried (e.g., using Na2SO4) and concentrated (e.g., in vacuo). The residue can then be chromatographed (e.g., over silica gel (Combiflash, 4 g column, 0-100%, hexanes-ethyl acetate, 11 minutes)) to provide Formula (I).

In some embodiments, Formula (I) (or any other formula recited above) can be recovered. Recovery can occur using any suitable method including but not limited to HPLC (e.g., reverse phase), LC, precipitation, centrifugation, column chromatography (e.g., size exclusion chromatography or ion exchange chromatography), use of silica gel, or combinations thereof.

In some embodiments, a method for the preparation of a compound of Formula (I) can comprise one or more of the above-mentioned steps. In certain embodiments, a method for preparing a compound of Formula (I) comprises(a) reacting a compound of Formula (II) with a compound of Formula (III) to result in a mixture comprising a compound of Formula (IV);(b) reacting a compound of Formula (IV) with a suitable compound (e.g., POCl3or POBr3) to convert an oxo to a halogen (e.g., F, Cl, Br, or I) to result in a mixture comprising a compound of Formula (V);(c) reacting a compound of Formula (V) with a compound of Formula (VI); and;(d) recovering Formula (I).

EXAMPLES

Example Set A

Synthetic Methods and Compound Characterization

Compound I-2 is synthesized according to the following synthetic scheme, which is one example of how molecules of Formula (I) can be synthesized.

The synthesis began by reacting commercially available 2-(chloromethyl)-4(3H)-quinazolinone (A) with secondary amine B. The resulting quinazolinone C was treated with phosphoryl chloride, which furnished chloroquinazoline D. Subsequent aromatic substitution with 3-methoxybenzylamine (E) afforded compound I-2.

Example Set B

Biological Characterization

Inhibition of Soft Agar Growth:

FIG. 1. Effects of some compounds of Formula (I) on RalGEF inhibitor on soft agar growth and normal growth in 2D of pancreatic carcinoma cell lines. InFIGS. 1A and 1B, Panc-1 (ATCC® CRL-1469™) and MiaPaCa-2 (ATCC® CRL-1420™) mutant Ras containing pancreatic tumor cell lines were plated in soft agar in the presence of 10 μM or 0.9 μM of compound I-1 and scored for the formation of colonies. InFIGS. 1C and 1D, compound I-1 was then tested for the ability to inhibit cell growth on the same cell lines under normal 2D growth conditions on plastic. “0” is the carrier. Cells were seeded at the same density and scored after two weeks in culture.

Human pancreatic tumor cell lines MiaPaca-2 and Panc-1 contain activated K-Ras and were used in the initial screen to identify compound I-1. Soft agar colony formation remains an established in vitro assay to predict tumorigenicity and was used to predict tumorigenicity for compound I-1. Quantitative assays showed that 10 μM of compound I-1 almost completely blocked the ability of the cells to grow in soft agar (FIGS. 1A and 1B). Titration experiments showed that compound I-1 exhibited an IC50 (i.e., a concentration that inhibits 50% of colony formation) of less than ˜1 μM (data not shown).

Several other compounds were tested against the Mia Paca-2 cell line using the soft agar colony formation in vitro assay in two experiments. The results for the two experiments are shown in Tables 2 and 3. For each table, DMSO was used as a control and, as such, within each table, the DMSO colonies are the measure against which each measurement in the corresponding table is compared.

Without being limited by theory, one of the advantages of RalGEFs as targets is that their activation can result in transformation, but their activation does not impact normal growth (e.g., in a plate-attached 2D culture). Therefore, according to this theory, a RalGEF inhibitor should block agar growth, but have little effect on normal 2D growth.

When we assayed compound I-1 against the same cell lines grown in 2D, we could detect no reduction in growth over the course of 1 week (FIGS. 1C and 1D). Compound I-2, has a similar effect to compound I-1 (data not shown).

RalGEFS in pancreatic cancer cell transformation:FIG. 2. Inhibition of multiple RalGEFs can sometimes have a synergistic inhibitory effect on growth in agar of pancreatic carcinoma cells. We identified an shRNA that was effective against RalGDS and made a stable matched pair of MiaPaCa-2 cells lines (FIG. 2A). We then transiently transfected the matched pair with validated siRNA against RGL2 (Invitrogen). Soft agar growth inhibition was then measured for the set of 4 cell systems. No effect on normal cell growth in 2D culture was observed (data not shown).

MiaPaCa-2 cells express only the RalGDS and RGL2 GEF family members. Stable shRNA inhibition of RGL2 inhibits the ability of the cells to grow in soft agar and form tumors in mice. We have now generated Mia-PaCa2 cells stably transfected with shRNA against RalGDS that are partially knocked down (FIG. 2A). We then transiently transfected the matched pair +/− shRNA-RalGDS with a pre-validated siRNA against RGL2 to simulate the effects of a pan-RalGEF inhibitor.FIG. 2Bshows that single RalGEF knockdown inhibited colony formation but that suppressing both RalGEFs had a greater than additive effect. Thus, blocking the action of multiple RalGEFs can sometimes be more effective than blocking a single RalGEF. Therefore, a small molecule that can act as a pan-RalGEF inhibitor could be effective.

Inhibition of Ras/RalGEF binding—FIG. 3. Some compounds of Formula (I) can be pan-RalGEF inhibitors, as shown by blocking the interaction of K-Ras with RalGDS and with RGL2. HEK-293 cells were co-transfected with HA-activated K-Ras and GFP-RalGDS (FIG. 3A) or RGL2 (FIG. 3B) in the presence of some compounds. The cells were lysed. The compound to be tested was added to the lysate to a final concentration 10 μM, which was then incubated with anti-GFP-beads overnight. The immunoprecipitate (IP) was then analyzed for the presence of Ras by immunoblot (IB).FIG. 3Ais an example of raw data.FIG. 3Bshows quantification of the RGL2 data with Compounds I-1 and I-2.

Compound I-1 interferes with the interaction of Ras and RalGEFs. HEK-293 cells were co-transfected with human influenza hemagglutinin (HA) tagged K-Ras 12v and GFP-tagged RalGDS or GFP-tagged RGL2 in the presence or absence of compound I-1. The cells were lysed and the degree of Ras/RalGEF interaction in each sample determined by immunoprecipitation (IP) followed by immunoblot (IB). Compound I-1 (but not carrier) inhibited the stable association of Ras and RalGDS (FIG. 3A). To determine if compound I-1 also inhibited a second RalGEF (RGL2), we performed similar experiments with RGL2.FIG. 3Bshows compound I-1 also inhibited RGL2. Thus, compound I-1 appears to be acting as a pan-RalGEF inhibitor. Compound I-2 was also tested using this assay; it was also found to inhibit RGL2 (FIG. 3B).

Specific Inhibition of endogenous Ras/RalGEF signaling.FIG. 4. Specific suppression of Ral activation by some compounds of Formula (I). Cells with endogenous hyper-activated Ras were assayed for endogenous Ral activation in the presence or absence of inhibitor (10 μM) using a Cell Biolabs (SanDiego Calif.) kit. Compound I-1 was dissolved in DMSO; DMSO serves as the carrier negative control. IP is immunoprecipitation. IB is immunoblot. Cell lysates were then assayed for the levels of MAPK pathway activation using a phosphoERK antibody. They were the same. Actin is a loading control.

RalGEFs drive Ral into the active, GTP bound form so it can bind its effector RalBP1. Inactive, GDP bound Ral does not bind RalBP1. Commercially available kits available from several vendors use RalBP1 attached to beads as an affinity reagent specific to the active form of Ral. By measuring the level of activated Ral and comparing it to the total levels of Ral, it is possible to measure the state of endogenous Ral activation. We assayed untransfected cells containing endogenous hyper-activated Ras for the effects of compound I-1 treatment on endogenous levels of active Ral levels. Compound I-1 suppressed the levels of endogenous active Ral (GTP bound) (FIG. 4). This effect was specific as no inhibition of the Ras/Raf/MAPK pathway activation was detected (e.g., levels of phospho ERK did not change).

Lack of in vivo toxicity for some compounds of Formula (I): Transgenic knockout studies and shRNA experiments on pancreatic cancer cells show that suppression of the Ras/RalGEF/Ral pathway inhibits the tumorigenic phenotype but does not have a severe impediment on normal cellular function. In tissue culture, compound I-1 had no effect on normal cell growth (FIG. 1) and neither did compound I-2 (data not shown). Preliminary in vivo studies show that doses of 25 mg/kg of compound I-1 have no obvious deleterious effect on mice (e.g., no behavior change and no weight loss) after ip injection over 2 weeks (data not shown).

Some compounds of Formula (I) suppress Ral GTP levels in vivo—FIG. 5NRG mice were provided with s.c. xenografts of Mia-PaCa2 cells. Tissue from the lungs of a carrier treated xenograft mouse and a compound I-1 treated mouse were assayed for the levels of activated RalA using a pull down assay kit using the manufacter's protocol (Cat. #BK040 from Cytoskeleton, Inc., Denver Colo.). The lung tissue from the treated xenograft animal shows reduced levels of active RalA. In particular,FIG. 5shows that compound I-1 appears to suppress the levels of active Ral bound to GTP in the lung tissue in vivo.

Some compounds of Formula (I) suppress pancreatic cancer cell metastasis—FIG. 6. NRG mice were sub-cutaneously inoculated with PBS (“no tumor cells”) or 5×106Ras driven Mia-PaCa-2 cells. Mice were allowed to recover for 1 week and then injected (ip) with PBS/DMSO carrier or 10 mg/kg of compound I-1 every other day for 2 weeks. One week later, mice were scored for primary tumor growth and lung metastasis. Effects on the primary tumor arising from the sub-cutaneous xenograft (which can be poorly vascularized and sometimes do not take up drugs well) were not obvious. However, metastasis was inhibited, as shown inFIG. 6A. Quantification is shown inFIG. 6B.

The headings used in the disclosure are not meant to suggest that all disclosure relating to the heading is found within the section that starts with that heading. Disclosure for any subject may be found throughout the specification.

As used in the disclosure, “a” or “an” means one or more than one, unless otherwise specified. As used in the claims, when used in conjunction with the word “comprising” the words “a” or “an” means one or more than one, unless otherwise specified. As used in the disclosure or claims, “another” means at least a second or more, unless otherwise specified. As used in the disclosure, the phrases “such as”, “for example”, and “e.g.” mean “for example, but not limited to” in that the list following the term (“such as”, “for example”, or “e.g.”) provides some examples but the list is not necessarily a fully inclusive list. The word “comprising” means that the items following the word “comprising” may include additional unrecited elements or steps; that is, “comprising” does not exclude additional unrecited steps or elements.

Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein (even if designated as preferred or advantageous) are not to be interpreted as limiting, but rather are to be used as an illustrative basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.