# Patent

## Title
Saturated acyl guanidine for inhibition of F1F0-ATPase

## Abstract

The invention provides saturated acyl guanidine compounds that inhibit F1F0-ATPase, and methods of using saturated acyl guanidine compounds as therapeutic agents to treat medical disorders, such as an immune disorder, inflammatory condition, or cancer.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a national stage of International (PCT) Patent Application Serial No. PCT/US2013/044736, filed Jun. 7, 2013, and published under PCT Article 21(2) in English, which claims the benefit of and priority to U.S. Provisional Patent Application Ser. No. 61/657,440, filed Jun. 8, 2012, the contents of each of which are hereby incorporated by reference.


FIELD OF THE INVENTION
The invention provides inhibitors of F1F0-ATPases (e.g., mitochondrial F1F0-ATPases) and their therapeutic use. In particular, the invention provides saturated acyl guanidine compounds that inhibit F1F0-ATPase, and methods of using saturated acyl guanidine compounds as therapeutic agents to treat a number of medical conditions.
BACKGROUND
Multicellular organisms exert precise control over cell number. A balance between cell proliferation and cell death achieves this homeostasis. Cell death occurs in nearly every type of vertebrate cell via necrosis or through a suicidal form of cell death, known as apoptosis. Apoptosis is triggered by a variety of extracellular and intracellular signals that engage a common, genetically programmed death mechanism.
Multicellular organisms use apoptosis to instruct damaged or unnecessary cells to destroy themselves for the good of the organism. Control of the apoptotic process therefore is very important to normal development, for example, fetal development of fingers and toes requires the controlled removal, by apoptosis, of excess interconnecting tissues, as does the formation of neural synapses within the brain. Similarly, controlled apoptosis is responsible for the sloughing off of the inner lining of the uterus (the endometrium) at the start of menstruation. While apoptosis plays an important role in tissue sculpting and normal cellular maintenance, it is also a component of the primary defense against cells and invaders (e.g., viruses) which threaten the well being of the organism.
Not surprisingly many diseases are associated with dysregulation of apoptotic cell death. Experimental models have established a cause-effect relationship between aberrant apoptotic regulation and the pathagenicity of various neoplastic, autoimmune and viral diseases. For instance, in the cell-mediated immune response, effector cells (e.g., cytotoxic T lymphocytes “CTLs”) destroy virus-infected cells by inducing the infected cells to undergo apoptosis. The organism subsequently relies on the apoptotic process to destroy the effector cells when they are no longer needed. Autoimmunity is normally prevented by the CTLs inducing apoptosis in each other and even in themselves. Defects in this process are associated with a variety of immune diseases such as lupus erythematosus and rheumatoid arthritis.
Multicellular organisms also use apoptosis to instruct cells with damaged nucleic acids (e.g., DNA) to destroy themselves prior to becoming cancerous. Some cancer-causing viruses overcome this safeguard by reprogramming infected (transformed) cells to abort the normal apoptotic process. For example, several human papilloma viruses (HPVs) have been implicated in causing cervical cancer by suppressing the apoptotic removal of transformed cells by producing a protein (E6) which inactivates the p53 apoptosis promoter. Similarly, the Epstein-Barr virus (EBV), the causative agent of mononucleosis and Burkitt's lymphoma, reprograms infected cells to produce proteins that prevent normal apoptotic removal of the aberrant cells thus allowing the cancerous cells to proliferate and to spread throughout the organism.
Still other viruses destructively manipulate a cell's apoptotic machinery without directly resulting in the development of a cancer. For example, destruction of the immune system in individuals infected with the human immunodeficiency virus (HIV) is thought to progress through infected CD4+ T cells (about 1 in 100,000) instructing uninfected sister cells to undergo apoptosis.
Some cancers that arise by non-viral means have also developed mechanisms to escape destruction by apoptosis. Melanoma cells, for instance, avoid apoptosis by inhibiting the expression of the gene encoding Apaf-1. Other cancer cells, especially lung and colon cancer cells, secrete high levels of soluble decoy molecules that inhibit the initiation of CTL mediated clearance of aberrant cells. Faulty regulation of the apoptotic machinery has also been implicated in various degenerative conditions and vascular diseases.
Controlled regulation of the apoptotic process and its cellular machinery is important to the survival of multicellular organisms. Typically, the biochemical changes that occur in a cell instructed to undergo apoptosis occur in an orderly procession. However, as shown above, flawed regulation of apoptosis can cause serious deleterious effects in the organism.
The need exists for improved compositions and methods for regulating the apoptotic processes in subjects afflicted with diseases and conditions characterized by faulty regulation of these processes (e.g., viral infections, hyperproliferative autoimmune disorders, chronic inflammatory conditions, and cancers). The present invention addresses this need and provides other related advantages.
SUMMARY
The invention provides saturated acyl guanidine compounds that inhibit F1F0-ATPase (e.g., mitochondrial F1F0-ATPase), pharmaceutical compositions comprising saturated acyl guanidine compounds, and methods of using such compounds and pharmaceutical compositions to treat a number of medical conditions. Accordingly, one aspect of the invention provides a family of compounds represented by Formula I:




including all stereoisomers, geometric isomers, and tautomers; or a pharmaceutically acceptable salt or solvate of any of the foregoing; wherein the variables are as defined in the detailed description. Additional saturated acyl guanidine compounds, e.g., compounds represented by Formula I-A, Formula I-A1, Formula II, Formula II-A, and Formula II-A1, are described in the detailed description. The foregoing compounds can be present in a pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier.

Another aspect of the invention provides a method of treating a subject suffering from a medical disorder. The method comprises administering to the subject a therapeutically effective amount of one or more saturated acyl guanidine compounds described herein, e.g., a compound of Formula I, I-A, I-A1, II, II-A, or II-A1, in order to ameliorate a symptom of the disorder. A large number of disorders can be treated using the saturated acyl guanidine compounds described herein. For example, the compounds described herein can be used to treat an immune disorder or inflammatory disorder, such as rheumatoid arthritis, psoriasis, chronic graft-versus-host disease, acute graft-versus-host disease, Crohn's disease, inflammatory bowel disease, multiple sclerosis, systemic lupus erythematosus, Celiac Sprue, idiopathic thrombocytopenic thrombotic purpura, myasthenia gravis, Sjogren's syndrome, scleroderma, ulcerative colitis, asthma, epidermal hyperplasia, and other medical disorders described herein. The compounds described herein can also be used to treat a cardiovascular disease, myeloma, lymphoma, cancer, or bacterial infection.
Another aspect of the invention provides a method of inhibiting a F1F0-ATPase, for example, a mitochondrial F1F0-ATPase. The method comprises exposing the F1F0-ATPase to a compound described herein, such as a compound Formula I, I-A, I-A1, II, II-A, or II-A1, to inhibit said F1F0-ATPase.


DETAILED DESCRIPTION OF THE INVENTION
The invention provides saturated acyl guanidine compounds that inhibit F1F0-ATPase (e.g., mitochondrial F1F0-ATPase), pharmaceutical compositions comprising the saturated acyl guanidine compounds, and methods of using the saturated acyl guanidine compounds and pharmaceutical compositions in therapy.
Exemplary compositions and methods of the present invention are described in more detail in the following sections: I. Modulators of F1F0-ATPase Activity; II. Saturated Acyl Guanidine Compounds; III. Therapeutic Applications of Saturated Acyl Guanidine Compounds, and IV. Pharmaceutical Compositions, Formulations, and Exemplary Administration Routes and Dosing Considerations. Aspects of the invention described in one particular section are not to be limited to any particular section.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as “Comprehensive Organic Synthesis” (B. M. Trost & I. Fleming, eds., 1991-1992); “Molecular cloning: a laboratory manual” Second Edition (Sambrook et al., 1989); “Oligonucleotide synthesis” (M. J. Gait, ed., 1984); “Animal cell culture” (R. I. Freshney, ed., 1987); the series “Methods in enzymology” (Academic Press, Inc.); “Handbook of experimental immunology” (D. M. Weir & C. C. Blackwell, eds.); “Gene transfer vectors for mammalian cells” (J. M. Miller & M. P. Calos, eds., 1987); “Current protocols in molecular biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: the polymerase chain reaction” (Mullis et al., eds., 1994); and “Current protocols in immunology” (J. E. Coligan et al., eds., 1991), each of which is herein incorporated by reference in its entirety.
To facilitate an understanding of the present invention, a number of terms and phrases are defined below.
As used herein, the term “guanidine” refers to a compound having the following core structure




including pharmaceutically acceptable salt forms.

The term “alkyl” refers to a saturated straight or branched hydrocarbon, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C1-C12alkyl, C1-C10alkyl, and C1-C6alkyl, respectively. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, etc.
The term “alkylene” refers to a diradical of an alkyl group. Exemplary alkylene groups include —CH2—, —CH2CH2—, and —CH2CH2CH2—.
The term “haloalkyl” refers to an alkyl group that is substituted with at least one halogen. For example, —CH2F, —CHF2, —CF3, —CH2CF3, —CF2CF3, and the like.
The term “hydroxyalkyl” refers to an alkyl group that is substituted with at least one hydroxyl group. In certain embodiments, the hydroxyalkyl group is an alkyl group that is substituted with one hydroxyl group.
The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as “C4-8 cycloalkyl,” derived from a cycloalkane. Exemplary cycloalkyl groups include cyclohexyl, cyclopentyl, cyclobutyl, and cyclopropyl.
The term “aralkyl” refers to an alkyl group substituted with an aryl group.
The term “heteroaralkyl” refers to an alkyl group substituted with a heteroaryl group.
The term “alkenyl” refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12alkenyl, C2-C10alkenyl, and C2-C6alkenyl, respectively. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, 4-(2-methyl-3-butene)-pentenyl, etc.
The term “alkynyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched group of 2-12, 2-8, or 2-6 carbon atoms, referred to herein as C2-C12alkynyl, C2-C8alkynyl, and C2-C6alkynyl, respectively. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl, 4-propyl-2-pentynyl, and 4-butyl-2-hexynyl, etc.
The term “aryl” is art-recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. Unless specified otherwise, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, —C(O)alkyl, —CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, heteroaryl, —CF3, —CN, or the like. The term “aryl” also includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic, and the other ring(s) may be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, and/or aryl. In certain embodiments, the aromatic group is not substituted, i.e., it is unsubstituted.
The terms “heterocyclyl” or “heterocyclic group” are art-recognized and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, alternatively 3- to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. Heterocycles may also be mono-, bi-, or other multi-cyclic ring systems. A heterocycle may be fused to one or more aryl, partially unsaturated, or saturated rings. Heterocyclyl groups include, for example, biotinyl, chromenyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl, dithiazolyl, homopiperidinyl, imidazolidinyl, isoquinolyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, oxolanyl, oxazolidinyl, phenoxanthenyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrazolinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolyl, thiazolidinyl, thiolanyl, thiomorpholinyl, thiopyranyl, xanthenyl, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, 2,3-dihydrobenzo[d]oxazolyl, and the like. Unless specified otherwise, the heterocyclic ring is optionally substituted at one or more positions with substituents such as alkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl and thiocarbonyl. In certain embodiments, the heterocyclcyl group is not substituted, i.e., it is unsubstituted.
The term “heteroaryl” is art-recognized and refers to aromatic groups that include at least one ring heteroatom. In certain instances, a heteroaryl group contains 1, 2, 3, or 4 ring heteroatoms. Representative examples of heteroaryl groups includes pyrrolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like. Unless specified otherwise, the heteroaryl ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, —C(O)alkyl, —CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl, —CF3, —CN, or the like. The term “heteroaryl” also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is heteroaromatic, and the other ring(s) may be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, and/or aryl. Exemplary heteroaryls that have a bicyclic ring system in which two carbon atoms are common to the adjoining ring include, for example, indazolyl, indolyl, and pyrazolo[3,4-c]pyridinyl.
The terms ortho, meta and para are art-recognized and refer to 1,2-, 1,3- and 1,4-disubstituted benzenes, respectively. For example, the names 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.
The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that may be represented by the general formula:




wherein R50 and R51 each independently represent hydrogen, alkyl, alkenyl, or —(CH2)m—R61; or R50 and R51, taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; wherein R61 is aryl, cycloalkyl, cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In certain embodiments, R50 and R51 each independently represent hydrogen or alkyl.

The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH2)m—R61, where m and R61 are described above.
The term “amide” or “amido” as used herein refers to a radical of the form —RaC(O)N(Rb)—, —RaC(O)N(Rb)Rc—, —C(O)NRbRc, or —C(O)NH2, wherein Ra, Rb and Rc are each independently selected from alkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, and nitro. The amide can be attached to another group through the carbon, the nitrogen, Rb, Rc, or Ra. The amide also may be cyclic, for example Rb and Rc, Ra and Rb, or Ra and Rc may be joined to form a 3- to 12-membered ring, such as a 3- to 10-membered ring or a 5- to 6-membered ring. The term “carboxamido” refers to the structure —C(O)NRbRc.
The term “sulfonamide” or “sulfonamido” as used herein refers to a radical having the structure —N(Rr)—S(O)2—Rs— or —S(O)2—N(Rr)Rs, where Rr, and Rs can be, for example, hydrogen, alkyl, aryl, cycloalkyl, and heterocyclyl. Exemplary sulfonamides include alkylsulfonamides (e.g., where Rs is alkyl), arylsulfonamides (e.g., where Rs is aryl), cycloalkyl sulfonamides (e.g., where Rs is cycloalkyl), and heterocyclyl sulfonamides (e.g., where Rs is heterocyclyl), etc.
The term “sulfonyl” as used herein refers to a radical having the structure RuSO2—, where Ru can be alkyl, aryl, cycloalkyl, and heterocyclyl, e.g., alkylsulfonyl. The term “alkylsulfonyl” as used herein refers to an alkyl group attached to a sulfonyl group.
The symbol “” indicates a point of attachment.
The compounds of the disclosure may contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers (such as enantiomers, mixtures of enantiomers, diastereomers, and mixtures of diastereomers) and geometric isomers (e.g., an E-isomer, Z-isomer, or mixture of E- and Z-isomers). These compounds may be designated by the symbols “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom. Stereoisomers include enantiomers, mixtures of enantiomers, diastereomers, and mixtures of diastereomers. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. Unless indicated otherwise, generic chemical structures and graphical representations of specific compounds encompass all stereoisomers and geometric isomers.
Individual stereoisomers of compounds of the present invention can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, or (3) direct separation of the mixture of optical enantiomers on chiral chromatographic columns. Stereoisomeric mixtures can also be resolved into their component stereoisomers by well known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Stereoisomers can also be obtained from stereomerically-pure intermediates, reagents, and catalysts by well known asymmetric synthetic methods.
As indicated above, the invention encompasses the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond or arrangement of substituents around a carbocyclic ring. Substituents around a carbon-carbon double bond are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the “E” and “Z” isomers.
Substituents around a carbon-carbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond. The arrangement of substituents around a carbocyclic ring are designated as “cis” or “trans.” The term “cis” represents substituents on the same side of the plane of the ring and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis/trans.”
Certain compounds described herein may exist as a single tautomer or as a mixture of tautomers. For example, certain guanidine compounds having a hydrogen atom attached to at least one of the guanidine nitrogen atoms can exist as a single tautomer or a mixture of tautomers. To illustrate, depending upon the substituents attached at the R1, R2 and R3 positions, the guanidine compound may exist as a single tautomer represented by A, B, or C, or as mixture of two or more of A, B, and C.




The compounds disclosed herein can exist in solvated as well as unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the invention embrace both solvated and unsolvated forms.
The invention also embraces isotopically labeled compounds of the invention which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F, and 36Cl, respectively.
Certain isotopically-labeled disclosed compounds (e.g., those labeled with 3H and 14C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., 3H) and carbon-14 (i.e., 14C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., 2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances.
Isotopically labeled compounds of the invention can generally be prepared by following procedures analogous to those disclosed in the e.g., Examples herein, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.
The term “IC50” is art-recognized and refers to the concentration of a compound that is required for 50% inhibition of its target.
The term “EC50” is art-recognized and refers to the concentration of a compound at which 50% of its maximal effect is observed.
The terms “subject” and “patient” refer to organisms to be treated by the methods of the present invention. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans. In the context of the invention, the terms “subject” and “patient” generally refer to an individual who will receive or who has received treatment (e.g., administration of a compound of the present invention and optionally one or more other agents) for a condition characterized by the dysregulation of apoptotic processes.
As used herein, the term “effective amount” refers to the amount of a compound sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. As used herein, the term “treating” includes any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.
The phrase “pathologically proliferating or growing cells” refers to a localized population of proliferating cells in an animal that is not governed by the usual limitations of normal growth.
As used herein, the term “un-activated target cell” refers to a cell that is either in the Go phase or one to which a stimulus has not been applied.
As used herein, the term “activated target lymphoid cell” refers to a lymphoid cell that has been primed with an appropriate stimulus to cause a signal transduction cascade, or alternatively, a lymphoid cell that is not in Go phase. Activated lymphoid cells may proliferate, undergo activation induced cell death, or produce one or more cytotoxins, cytokines, or other related membrane-associated proteins characteristic of the cell type (e.g., CD8+ or CD4+). They are also capable of recognizing and binding any target cell that displays a particular antigen on its surface, and subsequently releasing its effector molecules.
As used herein, the term “activated cancer cell” refers to a cancer cell that has been primed with an appropriate stimulus to cause signal transduction. An activated cancer cell may or may not be in the GO phase.
An activating agent is a stimulus that upon interaction with a target cell results in a signal transduction cascade. Examples of activating stimuli include, but are not limited to, small molecules, radiant energy, and molecules that bind to cell activation cell surface receptors. Responses induced by activation stimuli can be characterized by changes in, among others, intracellular Ca2+, superoxide, or hydroxyl radical levels; the activity of enzymes like kinases or phosphatases; or the energy state of the cell. For cancer cells, activating agents also include transforming oncogenes.
As used herein, the term “dysregulation of the process of cell death” refers to any aberration in the ability (e.g., predisposition) of a cell to undergo cell death via either necrosis or apoptosis. Dysregulation of cell death is associated with or induced by a variety of conditions, including for example, immune disorders (e.g., systemic lupus erythematosus, autoimmune disorders, rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, Sjögren's syndrome, etc.), chronic inflammatory conditions (e.g., psoriasis, asthma and Crohn's disease), hyperproliferative disorders (e.g., tumors, B cell lymphomas, T cell lymphomas, etc.), viral infections (e.g., herpes, papilloma, HIV), and other conditions such as osteoarthritis and atherosclerosis.
It should be noted that when the dysregulation is induced by or associated with a viral infection, the viral infection may or may not be detectable at the time dysregulation occurs or is observed. That is, viral-induced dysregulation can occur even after the disappearance of symptoms of viral infection.
A “hyperproliferative disorder,” as used herein refers to any condition in which a localized population of proliferating cells in an animal is not governed by the usual limitations of normal growth. Examples of hyperproliferative disorders include tumors, neoplasms, lymphomas and the like. A neoplasm is said to be benign if it does not undergo invasion or metastasis, and malignant if it does either of these. A metastatic cell or tissue means that the cell can invade and destroy neighboring body structures. Hyperplasia is a form of cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. Metaplasia is a form of controlled cell growth in which one type of fully differentiated cell substitutes for another type of differentiated cell. Metaplasia can occur in epithelial or connective tissue cells. A typical metaplasia involves a somewhat disorderly metaplastic epithelium.
The pathological growth of activated lymphoid cells often results in an immune disorder or a chronic inflammatory condition. As used herein, the term “immune disorder” refers to any condition in which an organism produces antibodies or immune cells which recognize the organism's own molecules, cells or tissues. Non-limiting examples of immune disorders include autoimmune disorders, immune hemolytic anemia, immune hepatitis, Berger's disease or IgA nephropathy, Celiac Sprue, chronic fatigue syndrome, Crohn's disease, dermatomyositis, fibromyalgia, graft-versus-host disease, Grave's disease, Hashimoto's thyroiditis, idiopathic thrombocytopenia purpura, lichen planus, multiple sclerosis, myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis, scleroderma, Sjorgren syndrome, systemic lupus erythematosus, type 1 diabetes, ulcerative colitis, vitiligo, tuberculosis, and the like.
As used herein, the term “chronic inflammatory condition” refers to a condition wherein the organism's immune cells are activated. Such a condition is characterized by a persistent inflammatory response with pathologic sequelae. This state is characterized by infiltration of mononuclear cells, proliferation of fibroblasts and small blood vessels, increased connective tissue, and tissue destruction. Examples of chronic inflammatory diseases include, but are not limited to, Crohn's disease, psoriasis, chronic obstructive pulmonary disease, inflammatory bowel disease, multiple sclerosis, and asthma. Immune diseases such as rheumatoid arthritis and systemic lupus erythematosus can also result in a chronic inflammatory state.
As used herein, the term “co-administration” refers to the administration of at least two agent(s) (e.g., a compound of the present invention) or therapies to a subject. In some embodiments, the co-administration of two or more agents/therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents/therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents/therapies are co-administered, the respective agents/therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents/therapies lowers the requisite dosage of a known potentially harmful (e.g., toxic) agent(s).
As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. [1975].
As used herein, the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention which, upon administration to a subject, is capable of providing a compound of this invention or an active metabolite or residue thereof. As is known to those of skill in the art, “salts” of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.
Examples of bases include, but are not limited to, alkali metals (e.g., sodium) hydroxides, alkaline earth metals (e.g., magnesium), hydroxides, ammonia, and compounds of formula NW4+, wherein W is C1-4 alkyl, and the like.
Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, NH4+, and NW4+ (wherein W is a C1-4 alkyl group), and the like.
For therapeutic use, salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
As used herein, the term “modulate” refers to the activity of a compound (e.g., a compound of the present invention) to affect (e.g., to promote or retard) an aspect of cellular function, including, but not limited to, cell growth, proliferation, apoptosis, and the like.
The terms “a,” “an” and “the” as used herein mean “one or more” and include the plural unless the context is inappropriate.
Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.
I. Modulators of F1F0-ATPase Activity
In some embodiments, the present invention regulates F1F0-ATPase activity (e.g., mitochondrial F1F0-ATPase activity) through the exposure of cells to compounds of the present invention. In some embodiments, the compounds inhibit ATP synthesis and ATP hydrolysis. The effect of the compounds can be measured by detecting any number of cellular changes. For example, mitochondrial F1F0-ATPase activity and/or cell death may be assayed as described herein and in the art. In some embodiments, cell lines are maintained under appropriate cell culturing conditions (e.g., gas (CO2), temperature and media) for an appropriate period of time to attain exponential proliferation without density dependent constraints. Cell number and or viability are measured using standard techniques, such as trypan blue exclusion/hemo-cytometry, or an Alamar Blue or MTT dye conversion assay. Alternatively, the cell may be analyzed for the expression of genes or gene products associated with aberrations in apoptosis or necrosis.
In some embodiments, exposing the compounds of the present invention to a cell induces apoptosis. In some embodiments, the present invention induces apoptosis or arrest of cell proliferation through interacting with the mitochondrial F1F0-ATPase. In some embodiments, the compounds of the present invention inhibit mitochondrial F1F0-ATPase activity through binding the OSCP. In some embodiments, the compounds of the present invention bind the junction between the OSCP and the F1 subunit of the mitochondrial F1F0-ATPase. In some embodiments, the compounds of the present invention bind the F1 subunit. In certain embodiments, screening assays of the present invention permit detection of binding partners of the OSCP, F1, or OSCP/F1 junction.
In some embodiments, exposing a compound of the present invention to a cell induces apoptosis. In some embodiments, the present invention causes an initial increase in cellular ROS levels (e.g., O2−). In further embodiments, exposure of the compounds of the present invention to a cell causes an increase in cellular O2− levels. In still further embodiments, the increase in cellular O2− levels resulting from the compounds of the present invention is detectable with a redox-sensitive agent that reacts specifically with O2− (e.g., dihydroethidium (DHE)).
In some embodiments, the present invention causes a collapse of a cell's mitochondrial transmembrane potential (ΔΨm). In some embodiments, a collapse of a cell's mitochondrial ΔΨm resulting from the present invention is detectable with a mitochondria-selective potentiometric probe (e.g., 3,3′-Dihexyloxacarbocyanine iodide, DiOC6). In further embodiments, a collapse of a cell's mitochondrial ΔΨm resulting from the present invention occurs after an initial increase in cellular O2− levels.
In some embodiments, the present invention enables caspase activation. In other embodiments, the present invention causes the release of cytochrome c from mitochondria. In further embodiments, the present invention alters cystolic cytochrome c levels. In still other embodiments, altered cystolic cytochrome c levels resulting from the present invention are detectable by immunoblotting cytosolic fractions. In some embodiments, diminished cystolic cytochrome c levels resulting from the present invention are detectable after a period of time (e.g., 10 hours). In further preferred embodiments, diminished cystolic cytochrome c levels resulting from the present invention are detectable after 5 hours.
In other embodiments, the present invention causes the opening of the mitochondrial permeability transition pore. In some embodiments, the cellular release of cytochrome c resulting from the present invention is consistent with a collapse of mitochondrial ΔΨm. In still further embodiments, the present invention causes an increase in cellular O2− levels after a mitochondrial ΔΨm collapse and a release of cytochrome c. In further embodiments, a rise in cellular O2− levels is caused by a mitochondrial ΔΨm collapse and release of cytochrome c resulting from the present invention.
In other embodiments, the present invention causes cellular caspase activation. In some embodiments, caspase activation resulting from the present invention is measurable with a pan-caspase sensitive fluorescent substrate (e.g., FAM-VAD-fmk). In still further embodiments, caspase activation resulting from the present invention tracks with a collapse of mitochondrial ΔΨm. In other embodiments, the present invention causes an appearance of hypodiploid DNA. In some embodiments, an appearance of hypodiploid DNA resulting from the present invention is slightly delayed with respect to caspase activation.
In some embodiments, the molecular target for the present invention is found within mitochondria. In further embodiments, the molecular target of the present invention involves the mitochondrial ATPase. The primary sources of cellular ROS include redox enzymes and the mitochondrial respiratory chain (hereinafter MRC). In some embodiments, cytochrome c oxidase (complex IV of the MRC) inhibitors (e.g., NaN3) preclude a present invention dependent increase in cellular ROS levels. In other embodiments, the ubiquinol-cytochrome c reductase component of MRC complex III inhibitors (e.g., FK506) preclude a present invention dependent increase in ROS levels.
II. Saturated Acyl Guanidine Compounds
One aspect of the invention provides a family of compounds represented by Formula I:




including all stereoisomers, geometric isomers, and tautomers; or a pharmaceutically acceptable salt or solvate of any of the foregoing; wherein:

A1 is heteroaryl optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of phenyl, alkyl, haloalkyl, cycloalkyl, halogen, hydroxyl, hydroxyalkyl, and C1-6alkoxy, wherein the phenyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, hydroxyalkyl, C1-6alkoxy, and cyano;
R1 is alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, alkylene-O-alkyl, alkylene-cycloalkyl, alkylene-heterocycloalkyl, aralkyl, 2,3-dihydro-1H-inden-1-yl, 2,3-dihydro-1H-inden-2-yl, 1,2,3,4-tetrahydronaphthalen-1-yl, or 1,2,3,4-tetrahydronaphthalen-2-yl, wherein the cycloalkyl, heterocycloalkyl, aralkyl, 2,3-dihydro-1H-inden-1-yl, 2,3-dihydro-1H-inden-2-yl, 1,2,3,4-tetrahydronaphthalen-1-yl, and 1,2,3,4-tetrahydronaphthalen-2-yl are optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, cycloalkyl, hydroxyl, alkoxyl, and cyano;
R2 and R4 each represent independently hydrogen or alkyl;
R3 is one of the following:

    
    
        (i) alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkoxyl, —O—(C(R5)2)m-alkoxyl, —N(R6)(R7), heterocycloalkyl, —N(R6)C(O)-alkyl, —C(O)N(R6)(R7), —N(R6)C(O)N(R6)(R7), halogen, haloalkyl, and cyano; or
        (ii) cycloalkyl, —(C(R5)2)m-cycloalkyl, aryl, or aralkyl, each of which are optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, hydroxyalkyl, alkoxy, —S-alkyl, and cyano;
    
    


R5 represents independently for each occurrence hydrogen, alkyl, or cycloalkyl; or two occurrences of R5 attached to the same carbon atom are taken together with said carbon atom to form a saturated carbocylic ring;
R6 and R7 each represent independently for each occurrence hydrogen, alkyl, or cycloalkyl; or R6 and R7 are taken together with the nitrogen atom to which they are attached to form a 3 to 7 membered heterocyclic ring optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, and C1-6alkoxy; and
m is 1, 2, 3, 4, or 5.
Definitions of the variables in Formula I above encompass multiple chemical groups. The application contemplates embodiments where, for example, i) the definition of a variable is a single chemical group selected from those chemical groups set forth above, ii) the definition is a collection of two or more of the chemical groups selected from those set forth above, and iii) the compound is defined by a combination of variables in which the variables are defined by (i) or (ii), e.g., such as where A1 is heteroaryl substituted with 1, 2, or 3 substituents independently selected from the group consisting of alkyl and haloalkyl; R1 is alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, alkylene-O-alkyl, or alkylene-cycloalkyl; R2 and R4 are hydrogen; and R3 is alkyl.
In certain embodiments, A1 is heteroaryl optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of alkyl, haloalkyl, cycloalkyl, halogen, and hydroxyl. In certain other embodiments, A1 is heteroaryl substituted with 1 or 2 substituents independently selected from the group consisting of alkyl and haloalkyl. In certain other embodiments, A1 is pyrazolyl substituted with haloalkyl, or A1 is pyrazolyl substituted with phenyl, wherein the phenyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen and haloalkyl. In certain other embodiments, A1 is pyrazolyl substituted with 1 or 2 substituents independently selected from the group consisting of alkyl and haloalkyl.
In certain embodiments, A1 is represented by:




wherein R* is phenyl, alkyl, or haloalkyl, wherein the phenyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, and alkoxy.

In certain embodiments, A1 is represented by:




wherein R* is phenyl, alkyl, or haloalkyl, wherein the phenyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, and alkoxy.

In certain embodiments, A1 is represented by:




wherein R* is haloalkyl.

In certain embodiments, A1 is one of the following:




In certain embodiments, R1 is alkyl. In certain other embodiments, R1 is cycloalkyl alkylene-cycloalkyl, each of which is optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, hydroxyl, and alkoxyl. In certain other embodiments, R1 is alkylene-heterocycloalkyl optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, hydroxyl, and alkoxyl. In certain other embodiments, R1 is aralkyl optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, cycloalkyl, hydroxyl, alkoxyl, and cyano. In certain other embodiments, R1 is benzyl optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, and alkoxyl.
In certain embodiments, R2 is hydrogen. In certain embodiments, R2 is alkyl, such as methyl or ethyl. In certain embodiments, R4 is hydrogen. In certain embodiments, R4 is alkyl, such as methyl or ethyl. In certain embodiments, R2 and R4 are hydrogen.
In certain embodiments, R3 is alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkoxyl, —O—(C(R5)2)m-alkoxyl, —N(R6)(R7), heterocycloalkyl, —N(R6)C(O)-alkyl, —C(O)N(R6)(R), —N(R6)C(O)N(R6)(R), halogen, haloalkyl, and cyano. In certain other embodiments, R3 is alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkoxyl, and cycloalkyl. In certain other embodiments, R3 is alkyl or cycloalkyl. In certain other embodiments, R3 is cyclobutyl, cyclopentyl, cyclohexyl, or C2-6 alkyl. In certain other embodiments, R3 is C1-6 alkyl substituted by C1-6 alkoxyl. In certain other embodiments, R3 is alkyl, such as propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, or 2,2-dimethyl-1-propyl.
In certain embodiments, R3 is phenyl, benzyl, or —(C(R5)2)m-cycloalkyl, each of which are optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, hydroxyalkyl, alkoxy, —S-alkyl, and cyano. In certain embodiments, R3 is phenyl substituted with 1 or 2 substituents independently selected from the group consisting of chloro, fluoro, and trifluoromethyl.
In certain embodiments, R5 is hydrogen. In certain embodiments, R5 is methyl.
In certain embodiments, R6 is hydrogen. In certain embodiments, R6 is alkyl, such as methyl or ethyl. In certain embodiments, R7 is hydrogen. In certain embodiments, R7 is alkyl, such as methyl or ethyl. In certain embodiments, R6 and R7 are hydrogen.
In certain embodiments, m is 1 or 2.
Another aspect of the invention provides a family of compounds represented by Formula I-A:




including all stereoisomers, geometric isomers, and tautomers; or a pharmaceutically acceptable salt or solvate of any of the foregoing; wherein:
A1 is heteroaryl substituted with 1 or 2 substituents independently selected from the group consisting of alkyl, haloalkyl, and cycloalkyl; or A1 is heteroaryl substituted with phenyl, wherein the phenyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, and alkyl;
R1 is alkyl, cycloalkyl, heterocycloalkyl, alkylene-O-alkyl, or alkylene-cycloalkyl, wherein the cycloalkyl and heterocycloalkyl are optionally substituted by 1 or 2 substituents independently selected from the group consisting of halogen, haloalkyl, and alkyl;
R2 and R4 are hydrogen;
R3 is one of the following:

    
    
        (i) alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkoxyl, and —O—(C(R5)2)m-alkoxyl; or
        (ii) cycloalkyl, —(C(R5)2)m-cycloalkyl, aryl, or aralkyl, each of which are optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, hydroxyalkyl, and alkoxy;
    
    


R5 represents independently for each occurrence hydrogen, alkyl, or cycloalkyl;
R6 and R7 each represent independently for each occurrence hydrogen, alkyl, or cycloalkyl; and
m is 1, 2, or 3.
Definitions of the variables in Formula I-A above encompass multiple chemical groups. The application contemplates embodiments where, for example, i) the definition of a variable is a single chemical group selected from those chemical groups set forth above, ii) the definition is a collection of two or more of the chemical groups selected from those set forth above, and iii) the compound is defined by a combination of variables in which the variables are defined by (i) or (ii). Further, embodiments described above for Formula I, such as the definitions of variables associated with Formula I, are reiterated here for use in connection with Formula I-A as appropriate.
Another aspect of the invention provides a family of compounds represented by Formula I-A1:




including all stereoisomers, geometric isomers, and tautomers; or a pharmaceutically acceptable salt or solvate of any of the foregoing; wherein:
R1 is C1-6 alkyl, C1-6 haloalkyl, C3-6 cycloalkyl, C1-6 alkylene-O—C1-6 alkyl, C1-6 alkylene-C3-6 cycloalkyl, or C1-6 alkylene-heterocycloalkyl, wherein the cycloalkyl and heterocycloalkyl are optionally substituted by 1 or 2 substituents independently selected from the group consisting of halogen, haloalkyl, and alkyl;
R3 is one of the following:

    
    
        (i) C1-6 alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl and alkoxyl; or
        (ii) C3-6 cycloalkyl or —(CH2)m-cycloalkyl, each of which is optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkyl, hydroxyalkyl, and alkoxy;
    
    


R* is phenyl, alkyl, haloalkyl, or cycloalkyl; wherein the phenyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, and C1-6alkoxy; and
m is 1, 2, or 3.
Definitions of the variables in Formula I-A1 above encompass multiple chemical groups. The application contemplates embodiments where, for example, i) the definition of a variable is a single chemical group selected from those chemical groups set forth above, ii) the definition is a collection of two or more of the chemical groups selected from those set forth above, and iii) the compound is defined by a combination of variables in which the variables are defined by (i) or (ii), e.g., such as R1 is C1-6 alkyl or C3-6 cycloalkyl, R3 is C1-6 alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl and alkoxyl, and R* is haloalkyl.
In certain embodiments, R1 is C1-6 alkyl. In certain embodiments, R1 is C3-6 cycloalkyl or C1-6 alkylene-C3-6 cycloalkyl, wherein the cycloalkyl is optionally substituted by 1 or 2 substituents independently selected from the group consisting of halogen, haloalkyl, and alkyl.
In certain embodiments, R3 is C1-6 alkyl optionally substituted by alkoxyl.
In certain embodiments, R* is haloalkyl. In certain embodiments, R* is trifluoromethyl.
Another aspect of the invention provides a family of compounds represented by Formula II:




including all stereoisomers, geometric isomers, and tautomers; or a pharmaceutically acceptable salt or solvate of any of the foregoing; wherein:





wherein B2 is a 6-membered heteroaromatic group;

R1 is alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, alkylene-O-alkyl, alkylene-cycloalkyl, alkylene-heterocycloalkyl, aralkyl, 2,3-dihydro-1H-inden-1-yl, 2,3-dihydro-1H-inden-2-yl, 1,2,3,4-tetrahydronaphthalen-1-yl, or 1,2,3,4-tetrahydronaphthalen-2-yl; wherein the cycloalkyl, heterocycloalkyl, aralkyl, 2,3-dihydro-1H-inden-1-yl, 2,3-dihydro-1H-inden-2-yl, 1,2,3,4-tetrahydronaphthalen-1-yl, and 1,2,3,4-tetrahydronaphthalen-2-yl are optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, cycloalkyl, hydroxyl, alkoxyl, and cyano;
R2 and R4 each represent independently hydrogen or alkyl;
R3 is one of the following:

    
    
        (i) alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkoxyl, —O—(C(R5)2)m-alkoxyl, —N(R6)(R), heterocycloalkyl, —N(R6)C(O)-alkyl, —C(O)N(R6)(R7), —N(R6)C(O)N(R6)(R76)(R), halogen, haloalkyl, and cyano; or
        (ii) cycloalkyl, —(C(R5)2)m-cycloalkyl, aryl, or aralkyl, each of which are optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, hydroxyalkyl, alkoxy, —S-alkyl, and cyano;
    
    


R5 represents independently for each occurrence hydrogen, alkyl, or cycloalkyl; or two occurrences of R5 attached to the same carbon atom are taken together with said carbon atom to form a saturated carbocylic ring;
R6 and R7 each represent independently for each occurrence hydrogen, alkyl, or cycloalkyl; or R6 and R7 are taken together with the nitrogen atom to which they are attached to form a 3 to 7 membered heterocyclic ring optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, and C1-6alkoxy;
R8 is hydrogen, or R8 is alkyl substituted by hydroxyl, —N(H)(R6), heterocycloalkyl, —N(H)C(O)R10, —C(O)N(H)(R6), or —N(H)C(O)N(R6)(R);
R9 represents independently for each occurrence halogen, haloalkyl, alkyl, cycloalkyl, heterocycloalkyl, hydroxyl, C1-6alkoxy, or cyano;
R10 is alkyl, cycloalkyl, aryl, or aralkyl;
m is 1, 2, 3, 4, or 5; and
n is 0, 1, 2, 3, or 4.
Definitions of the variables in Formula II above encompass multiple chemical groups. The application contemplates embodiments where, for example, i) the definition of a variable is a single chemical group selected from those chemical groups set forth above, ii) the definition is a collection of two or more of the chemical groups selected from those set forth above, and iii) the compound is defined by a combination of variables in which the variables are defined by (i) or (ii), e.g., such as where A1 is




R1 is alkyl, haloalkyl, hydroxyalkyl, or cycloalkyl, R2 and R4 are hydrogen, and R3 is alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl and alkoxyl.

Accordingly, in certain embodiments, A1 is




It is understood that the chemical formula





conveys that the indazolyl structure shown can be attached to the guanidine core via (i) a carbon atom of the 5-membered ring of the indazolyl structure or (ii) a carbon atom of the 6-membered ring of the indazolyl structure. An example of where the indazolyl is attached to the guanidine core via a carbon atom of the 5-membered ring of the indazolyl structure is depicted below:





Examples of where the indazolyl is attached to the guanidine core via a carbon atom of the 6-membered ring of the indazolyl structure is depicted by the following formulae:




In certain other embodiments, A1 is




In certain embodiments, A1 is





In certain embodiments, A1 is




In certain other embodiments, A1 is





In certain embodiments, A1 is




In certain other embodiments, A1 is





In certain other embodiments, A1 is





In certain embodiments, A1 is




In certain other embodiments, A1 is





In certain other embodiments, A1 is





In certain embodiments, A1 is one of the following:




In certain embodiments, R1 is alkyl. In certain other embodiments, R1 is cycloalkyl or alkylene-cycloalkyl, each of which is optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, hydroxyl, and alkoxyl. In certain other embodiments, R1 is alkylene-heterocycloalkyl optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, hydroxyl, and alkoxyl. In certain other embodiments, R1 is aralkyl optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, cycloalkyl, hydroxyl, alkoxyl, and cyano. In certain other embodiments, R1 is benzyl optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, and alkoxyl.
In certain embodiments, R2 is hydrogen. In certain embodiments, R2 is alkyl, such as methyl or ethyl. In certain embodiments, R4 is hydrogen. In certain embodiments, R4 is alkyl, such as methyl or ethyl. In certain embodiments, R2 and R4 are hydrogen.
In certain embodiments, R3 is alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkoxyl, —O—(C(R5)2)m-alkoxyl, —N(R6)(R), heterocycloalkyl, —N(R6)C(O)-alkyl, —C(O)N(R6)(R7), —N(R6)C(O)N(R6)(R7), halogen, haloalkyl, and cyano. In certain other embodiments, R3 is alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkoxyl, and cycloalkyl. In certain other embodiments, R3 is alkyl or cycloalkyl. In certain other embodiments, R3 is cyclobutyl, cyclopentyl, cyclohexyl, or C2-6 alkyl. In certain other embodiments, R3 is C1-6 alkyl substituted by C1-6 alkoxyl. In certain other embodiments, R3 is alkyl, such as propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, or 2,2-dimethyl-1-propyl.
In certain embodiments, R3 is phenyl, benzyl, or —(C(R5)2)m-cycloalkyl, each of which are optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, hydroxyalkyl, alkoxy, —S-alkyl, and cyano. In certain embodiments, R3 is phenyl substituted with 1 or 2 substituents independently selected from the group consisting of chloro, fluoro, and trifluoromethyl.
In certain embodiments, R5 is hydrogen. In certain embodiments, R5 is methyl.
In certain embodiments, R6 is hydrogen. In certain embodiments, R6 is alkyl, such as methyl or ethyl. In certain embodiments, R7 is hydrogen. In certain embodiments, R7 is alkyl, such as methyl or ethyl. In certain embodiments, R6 and R7 are hydrogen.
In certain embodiments, R8 is hydrogen.
In certain embodiments, R9 represents independently for each occurrence halogen or haloalkyl. In certain other embodiments, R9 represents independently for each occurrence chloro, fluoro, or trifluoromethyl.
In certain embodiments, R10 is alkyl, such as methyl or ethyl.
In certain embodiments, n is 1 or 2. In certain embodiments, p is 1 or 2. In certain embodiments, m is 1 or 2.
In certain embodiments, A1 is




R8 is hydrogen; R9 represents independently for each occurrence halogen, haloalkyl, alkyl, cycloalkyl, heterocycloalkyl, hydroxyl, C1-6alkoxy, or cyano; and n is 0, 1, 2, or 3.

Another aspect of the invention provides a family of compounds represented by Formula II-A:




including all stereoisomers, geometric isomers, and tautomers; or a pharmaceutically acceptable salt or solvate of any of the foregoing; wherein:
A1 is




R1 is alkyl, cycloalkyl, heterocycloalkyl, alkylene-O-alkyl, or alkylene-cycloalkyl, wherein the cycloalkyl and heterocycloalkyl are optionally substituted by 1 or 2 substituents independently selected from the group consisting of halogen, haloalkyl, and alkyl;
R2 and R4 are hydrogen;
R3 is one of the following:

    
    
        (i) alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkoxyl, and —O—(C(R5)2)m-alkoxyl; or
        (ii) cycloalkyl, —(C(R5)2)m-cycloalkyl, aryl, or aralkyl, each of which are optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, hydroxyalkyl, and alkoxy;
    
    


R5 represents independently for each occurrence hydrogen, alkyl, or cycloalkyl;
R6 and R7 each represent independently for each occurrence hydrogen, alkyl, or cycloalkyl;
R8 is hydrogen;
R9 represents independently for each occurrence halogen, haloalkyl, alkyl, or cycloalkyl;
m is 1, 2, or 3; and
n is 1 or 2.
Definitions of the variables in Formula II-A above encompass multiple chemical groups. The application contemplates embodiments where, for example, i) the definition of a variable is a single chemical group selected from those chemical groups set forth above, ii) the definition is a collection of two or more of the chemical groups selected from those set forth above, and iii) the compound is defined by a combination of variables in which the variables are defined by (i) or (ii). Further, embodiments described above for Formula II, such as the definitions of variables associated with Formula II, are reiterated here for use in connection with Formula II-A as appropriate.
Another aspect of the invention provides a family of compounds represented by Formula II-A1:




including all stereoisomers, geometric isomers, and tautomers; or a pharmaceutically acceptable salt or solvate of any of the foregoing; wherein:
Y1, Y2, Y3, and Y4 each represent independently hydrogen, chloro, fluoro, hydroxyl, or —CF3; and
R1 is C1-6 alkyl, C1-6 haloalkyl, C3-6 cycloalkyl, C1-6 alkylene-O—C1-6 alkyl, C1-6 alkylene-C3-6 cycloalkyl, or C1-6 alkylene-heterocycloalkyl, wherein the cycloalkyl and heterocycloalkyl are optionally substituted by 1 or 2 substituents independently selected from the group consisting of halogen, haloalkyl, and alkyl;
R2 and R4 each represent independently hydrogen or alkyl;
R3 is one of the following:

    
    
        (i) C1-6 alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl and alkoxyl; or
        (ii) C3-6 cycloalkyl or —(CH2)m-cycloalkyl, each of which is optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkyl, hydroxyalkyl, and alkoxy; and
    
    


m is 1, 2, or 3.
Definitions of the variables in Formula II-A1 above encompass multiple chemical groups. The application contemplates embodiments where, for example, i) the definition of a variable is a single chemical group selected from those chemical groups set forth above, ii) the definition is a collection of two or more of the chemical groups selected from those set forth above, and iii) the compound is defined by a combination of variables in which the variables are defined by (i) or (ii), e.g., such as where R1 is C1-6 alkyl or C3-6 cycloalkyl, and R3 is C1-6 alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl and alkoxyl.
In certain embodiments, R1 is C1-6 alkyl. In certain embodiments, R1 is C3-6 cycloalkyl or C1-6 alkylene-C3-6 cycloalkyl, each of which is optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, hydroxyl, and alkoxyl.
In certain embodiments, R2 and R4 are hydrogen.
In certain embodiments, R3 is C1-6 alkyl optionally substituted by alkoxyl.
In certain embodiments, Y1, Y2, and Y4 are hydrogen; and Y3 is fluoro or —CF3.
In certain embodiments, Y1 and Y4 are hydrogen; and Y2 and Y3 are each independently fluoro or —CF3.
The description above for Formula I, I-A, I-A1, II, II-A, and II-A1 describes multiple embodiments providing definitions for variables used herein. The application specifically contemplates all combinations of such embodiments.
In certain embodiments, the compound is one of the compounds listed in any one of Tables 1-4 herein, or a pharmaceutically acceptable salt thereof. It is understood that the foregoing compounds can be combined with a pharmaceutically acceptable carrier to produce a pharmaceutical composition.







TABLE 1







PYRAZOLYL GUANIDINES.










No.
Compound







I-1 







 



I-2 







 



I-3 







 



I-4 







 



I-5 







 



I-6 







 



I-7 







 



I-8 







 



I-9 







 



I-10







 



I-11







 



I-12







 



I-13







 



I-14







 



I-15







 



I-16







 



I-17







 



I-18







 



I-19







 



I-20







 



I-21







 



I-22







 



I-23







 



I-24







 



I-25







 



I-26







 



I-27







 



I-28







 



I-29







 



I-30







 



I-31







 



I-32







 



I-33







 



I-34







 



I-35







 



I-36







 



I-37







 



I-38







 



I-39







 



I-40





















TABLE 2







ADDITIONAL PYRAZOLYL GUANIDINES.








 










No.
A
X
Y





II-1 








isobutyl


 


II-2 








sec-butyl


 


II-3 








tert-butyl


 


II-4 








ethyl


 


II-5 








propyl


 


II-6 








isopropyl


 


II-7 








—C(H)(Me)CH2OMe


 


II-8 








—CH2CH2OMe


 


II-9 








—CH2CH2OEt


 


II-10








cyclopropyl


 


II-11








cyclopentyl


 


II-12








cyclohexyl


 


II-13








1-methylcyclobutyl


 


II-14








1-methylcyclopentyl


 


II-15








4-methylcyclohexyl


 


II-16
—CF3




isobutyl


 


II-17
—CF3




sec-butyl


 


II-18
—CF3




tert-butyl


 


II-19
—CF3




ethyl


 


II-20
—CF3




propyl


 


II-21
—CF3




isopropyl


 


II-22
—CF3




—C(H)(Me)CH2OMe


 


II-23
—CF3




—CH2CH2OMe


 


II-24








—CH2CH2OEt


 


II-25








cyclopropyl


 


II-26








cyclopentyl


 


II-27








cyclohexyl


 


II-28








1-methylcyclobutyl


 


II-29








1-methylcyclopentyl


 


II-30








4-methylcyclohexyl


 


II-31








isobutyl


 


II-32








sec-butyl


 


II-33








isopropyl


 


II-34








—C(H)(Me)CH2OMe


 


II-35








—C(H)(Me)CH2OMe


 


II-36








isobutyl


 


II-37








sec-butyl


 


II-38








tert-butyl


 


II-39








isopropyl


 


II-40








—C(H)(Me)CH2OMe


 


II-41














 


II-42














 


II-43
—CF3










 


II-44














 


II-45




























TABLE 3







INDAZOLYL GUANIDINES.










No.
Compound







III-1 







 



III-2 







 



III-3 







 



III-4 







 



III-5 







 



III-6 







 



III-7 







 



III-8 







 



III-9 







 



III-10







 



III-11







 



III-12







 



III-13







 



III-14







 



III-15







 



III-16







 



III-17







 



III-18







 



III-19







 



III-20







 



III-21







 



III-22







 



III-23







 



III-24







 



III-25







 



III-26







 



III-27







 



III-28







 



III-29







 



III-30







 



III-31







 



III-32







 



III-33







 



III-34







 



III-35







 



III-36





















TABLE 4







ADDITIONAL INDAZOLYL GUANIDINES.








 










No.
A
X
Y





IV-1 








isobutyl


 


IV-2 








sec-butyl


 


IV-3 








tert-butyl


 


IV-4 








ethyl


 


IV-5 








propyl


 


IV-6 








isopropyl


 


IV-7 








—C(H)(Me)CH2OMe


 


IV-8 








—CH2CH2OMe


 


IV-9 








—CH2CH2OEt


 


IV-10








cyclopropyl


 


IV-11








cyclopentyl


 


IV-12








cyclohexyl


 


IV-13








1-methylcyclobutyl


 


IV-14








1-methylcyclopentyl


 


IV-15








4-methylcyclohexyl


 


IV-16








isobutyl


 


IV-17








sec-butyl


 


IV-18








tert-butyl


 


IV-19








ethyl


 


IV-20








propyl


 


IV-21








isopropyl


 


IV-22
—CF3




—C(H)(Me)CH2OMe


 


IV-23
—CF3




—CH2CH2OMe


 


IV-24
—CF3




—CH2CH2OEt


 


IV-25
—CF3




cyclopropyl


 


IV-26
—CF3




cyclopentyl


 


IV-27
—CF3




cyclohexyl


 


IV-28
—CF3




1-methylcyclobutyl


 


IV-29








1-methylcyclopentyl


 


IV-30








4-methylcyclohexyl


 


IV-31








isobutyl


 


IV-32








sec-butyl


 


IV-33








isopropyl


 


IV-34








—C(H)(Me)CH2OMe


 


IV-35








—C(H)(Me)CH2OMe


 


IV-36








isobutyl


 


IV-37








sec-butyl


 


IV-38








tert-butyl


 


IV-39








isopropyl


 


IV-40








—C(H)(Me)CH2OMe


 


IV-41














 


IV-42














 


IV-43














 


IV-44














 


IV-45














 


IV-46








isobutyl


 


IV-47








sec-butyl


 


IV-48








isopropyl


 


IV-49








—C(H)(Me)CH2OMe


 


IV-50








—C(H)(Me)CH2OMe


 


IV-51








isobutyl


 


IV-52








1-methylcyclopentyl


 


IV-53
—CCF3




cyclohexyl


 


IV-54














 


IV-55





















Exemplary methods for preparing compounds described herein are provided in the examples. Further exemplary procedures for making various compounds described herein are described in Scheme 1 below. The synthetic scheme is provided for the purpose of illustrating the invention, but not for limiting the scope or spirit of the invention. Starting materials can be obtained from commercial sources or be prepared based on procedures described in the literature.
The synthetic route in Scheme 1 involves reacting an optionally substituted saturated acyl chloride with potassium thiocyanate to form an acyl isothiocyanate intermediate. This acyl isothiocyanate intermediate is treated with a first amine (A1-NH2) to form an acyl thiourea. The acyl thiourea is reacted with 1-ethyl-2′,2′-dimethylaminopropylcarbodiimide (EDC) and a second amine compound (e.g., R3—NH2) to form the desired saturated acyl guanidine compound. To the extent either the first amine or the second amine compound contain a further functional group that may undergo reaction under the conditions illustrated in Scheme 1, standard protecting group strategies for protection and deprotection may be employed. See, for example, Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991.




where variables R1, A1, and R3 are, for example, as defined in association with Formula I above.

III. Therapeutic Applications of Saturated Acyl Guanidine Compounds

It is contemplated that the guanidine compounds described herein, such as the guanidine compounds of Formula I, I-A, I-A1, II, II-A, or II-A1, provide therapeutic benefits to patients suffering from any one or more of a number of conditions, e.g., diseases characterized by dysregulation of F1F0-ATPase activity, diseases characterized by dysregulation of necrosis and/or apoptosis processes in a cell or tissue, disease characterized by aberrant cell growth and/or hyperproliferation. The compounds described herein can also be used to treat a variety of dysregulatory disorders related to cellular death as described elsewhere herein. Additionally, the compounds described herein can be used to inhibit ATP synthesis.
Accordingly, one aspect of the invention provides a method of treating a subject suffering from a medical disorder. The method comprises administering to the subject a therapeutically effective amount of one or more saturated acyl guanidine compounds described herein, e.g., a compound of Formula I, I-A, I-A1, II, II-A, or II-A1, as described in Section II above, in order to ameliorate a symptom of the disorder.
A large number of medical disorders can be treated using the guanidine compounds described herein. For example, the compounds described herein can be used to treat medical disorders characterized by dysregulation of necrosis and/or apoptosis processes in a cell or tissue, diseases characterized by aberrant cell growth and/or hyperproliferation, etc., or lupus, rheumatoid arthritis, psoriasis, chronic graft-versus-host disease, acute graft-versus-host disease, Crohn's disease, inflammatory bowel disease, multiple sclerosis, cardiovascular disease, myeloma, lymphoma, cancer, and bacterial infection. In certain embodiments, the cancer is a solid tumor, leukemia, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, stomach cancer, cervical cancer, testicular tumor, skin cancer, rectal cancer, thyroid cancer, kidney cancer, uterus cancer, esophagus cancer, liver cancer, an acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, or retinoblastoma.
Although not wishing to be bound to a particular theory, it is believed that the compounds impart therapeutic benefit by modulating (e.g., inhibiting) the activity of the F1F0-ATPase complexes (e.g., mitochondrial F1F0-ATPase complexes) in affected cells or tissues. In some embodiments, the compositions of the present invention are used to treat immune/chronic inflammatory conditions (e.g., psoriasis, autoimmune disorders, organ-transplant rejection, and epidermal hyperplasia). In further embodiments, the compositions of the present invention are used in conjunction with stenosis therapy to treat compromised (e.g., occluded) vessels.
In certain embodiments, a composition comprising a guanidine compound is administered under conditions (e.g., timing, dose, co-administration with other agent, mode of administration, selection of subject, use of targeting agents, etc.) that maximize desired effects directed at the F1F0-ATPase.
In certain embodiments, the medical disorder is an immune disorder. In certain other embodiments, the medical disorder is an inflammatory disorder. In certain other embodiments, the medical disorder is an autoimmune disorder. In certain other embodiments, the medical disorder is rheumatoid arthritis, psoriasis, chronic graft-versus-host disease, acute graft-versus-host disease, Crohn's disease, inflammatory bowel disease, multiple sclerosis, systemic lupus erythematosus, Celiac Sprue, idiopathic thrombocytopenic thrombotic purpura, myasthenia gravis, Sjogren's syndrome, scleroderma, ulcerative colitis, asthma, uveitis, or epidermal hyperplasia.
In certain other embodiments, the medical disorder is cartilage inflammation, bone degradation, arthritis, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reter's Syndrome, SEA Syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reter's Syndrome, dermatomyositis, psoriatic arthritis, vasculitis, myolitis, polymyolitis, dermatomyolitis, osteoarthritis, polyarteritis nodossa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, dermatitis, atopic dermatitis, atherosclerosis, Still's disease, chronic obstructive pulmonary disease, Guillain-Barre disease, Type I diabetes mellitus, Graves' disease, Addison's disease, Raynaud's phenomenon, or autoimmune hepatitis. In certain embodiments, the psoriasis is plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, or erythrodermic psoriasis.
In certain other embodiments, the medical disorder is Crohn's disease, inflammatory bowel disease, multiple sclerosis, graft-versus-host disease, lupus, rheumatoid arthritis, or psoriasis. In certain other embodiments, the medical disorder is cardiovascular disease, myeloma, lymphoma, or cancer. In certain other embodiments, the medical disorder is lupus, rheumatoid arthritis, psoriasis, graft-versus-host disease, myeloma, or lymphoma. In certain other embodiments, the medical disorder is cardiovascular disease or cancer. In certain other embodiments, the medical disorder is Crohn's disease, inflammatory bowel disease, or multiple sclerosis. In certain other embodiments, the medical disorder is graft-versus-host disease. In further embodiments, the medical disorder is a bacterial infection. In certain embodiments, the patient (or subject) is a human.
As indicated above, the guanidine compounds described herein can be used in the treatment of a bacterial infection. A variety of bacteria are contemplated to be susceptible to the guanidine compounds. Representative bacteria include Staphylococci species, e.g., S. aureus; Enterococci species, e.g., E. faecalis and E. faecium; Streptococci species, e.g., S. pyogenes and S. pneumoniae; Escherichia species, e.g., E. coli, including enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic and enteroaggregative E. coli strains; Haemophilus species, e.g., H. influenza; and Moraxella species, e.g., M. catarrhalis. Other examples include Mycobacteria species, e.g., M. tuberculosis, M. avian-intracellulare, M. kansasii, M. bovis, M. africanum, M. genavense, M. leprae, M. xenopi, M. simiae, M. scrofulaceum, M. malmoense, M. celatum, M. abscessus, M. chelonae, M. szulgai, M. gordonae, M. haemophilum, M. fortuni and M. marinum; Corynebacteria species, e.g., C. diphtheriae; Vibrio species, e.g., V. cholerae; Campylobacter species, e.g., C. jejuni; Helicobacter species, e.g., H. pylori; Pseudomonas species, e.g., P. aeruginosa; Legionella species, e.g., L. pneumophila; Treponema species, e.g., T. pallidum; Borrelia species, e.g., B. burgdorferi; Listeria species, e.g., L monocytogenes; Bacillus species, e.g., B. cereus; Bordatella species, e.g., B. pertussis; Clostridium species, e.g., C. perfringens, C. tetani, C. difficile and C. botulinum; Neisseria species, e.g., N. meningitidis and N. gonorrhoeae; Chlamydia species, e.g., C. psittaci, C. pneumoniae and C. trachomatis; Rickettsia species, e.g., R. rickettsii and R. prowazekii; Shigella species, e.g., S. sonnei; Salmonella species, e.g., S. typhimurium; Yersinia species, e.g., Y. enterocolitica and Y. pseudotuberculosis; Klebsiella species, e.g., K. pneumoniae; Mycoplasma species, e.g., M. pneumoniae; and Trypanosoma brucei. In certain embodiments, the guanidine compounds described herein are used to treat a subject suffering from a bacterial infection selected from the group consisting of S. aureus, E. faecalis, E. faecium, S. pyogenes, S. pneumonia, and P. aeruginosa. In certain embodiments, the guanidine compounds described herein are used to treat a subject suffering from a Trypanosoma brucei infection.
The antibacterial activity of the compounds described herein may be evaluated using standard assays known in the art, such as the microbroth dilution minimum inhibition concentration (MIC) assay, as further described in National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing; Fourteenth Informational Supplement. NCCLS document M100-S14 {ISBN 1-56238-516-X}. This assay may be used to determine the minimum concentration of a compound necessary to prevent visible bacterial growth in a solution. In general, the drug to be tested is serially diluted into wells, and aliquots of liquid bacterial culture are added. This mixture is incubated under appropriate conditions, and then tested for growth of the bacteria. Compounds with low or no antibiotic activity (a high MIC) will allow growth at high concentrations of compound, while compounds with high antibiotic activity will allow bacterial growth only at lower concentrations (a low MIC).
The assay uses stock bacterial culture conditions appropriate for the chosen strain of bacteria. Stock cultures from the permanent stock culture collection can be stored as frozen suspensions at −70° C. Cultures may be suspended in 10% skim milk (BD) prior to snap freezing in dry ice/ethanol and then placed in a −70° C. freezer. Cultures may be maintained on Tryptic Soy Agar containing 5% Sheep Blood at room temperature (20° C.), and each culture may be recovered from frozen form and transferred an additional time before MIC testing. Fresh plates are inoculated the day before testing, incubated overnight, and checked to confirm purity and identity.
The identity and purity of the cultures recovered from the stock culture can be confirmed to rule out the possibility of contamination. The identity of the strains may be confirmed by standard microbiological methods (See, e.g., Murray et al., Manual of Clinical Microbiology, Eighth Edition. ASM Press {ISBN 1-55581-255-4}). In general, cultures are streaked onto appropriate agar plates for visualization of purity, expected colony morphology, and hemolytic patterns. Gram stains can also be utilized. The identities are confirmed using a MicroScan WalkAway 40 SI Instrument (Dade Behring, West Sacramento, Calif.). This device utilizes an automated incubator, reader, and computer to assess for identification purposes the biochemical reactions carried out by each organism. The MicroScan WalkAway can also be used to determine a preliminary MIC, which may be confirmed using the method described below.
Frozen stock cultures may be used as the initial source of organisms for performing microbroth dilution minimum inhibition concentration (MIC) testing. Stock cultures are passed on their standard growth medium for at least 1 growth cycle (18-24 hours) prior to their use. Most bacteria may be prepared directly from agar plates in 10 mL aliquots of the appropriate broth medium. Bacterial cultures are adjusted to the opacity of a 0.5 McFarland Standard (optical density value of 0.28-0.33 on a Perkin-Elmer Lambda EZ150 Spectrophotometer, Wellesley, Mass., set at a wavelength of 600 nm). The adjusted cultures are then diluted 400 fold (0.25 mL inoculum+100 mL broth) in growth media to produce a starting suspension of approximately 5×105 colony forming units (CFU)/mL. Most bacterial strains may be tested in cation adjusted Mueller Hinton Broth (CAMHB).
Test compounds (“drugs”) are solubilized in a solvent suitable for the assay, such as DMSO. Drug stock solutions may be prepared on the day of testing. Microbroth dilution stock plates may be prepared in two dilution series, 64 to 0.06 μg drug/mL and 0.25 to 0.00025 μg drug/mL. For the high concentration series, 200 μL of stock solution (2 mg/mL) is added to duplicate rows of a 96-well microtiter plate. This is used as the first well in the dilution series. Serial two-fold decremental dilutions are made using a BioMek FX robot (Beckman Coulter Inc., Fullerton, Calif.) with 10 of the remaining 11 wells, each of which will contain 100 μL of the appropriate solvent/diluent. Row 12 contains solvent/diluent only and serves as the control. For the first well of the low concentration series, 200 μL of an 8 μg/mL stock are added to duplicate rows of a 96-well plate. Serial two-fold dilutions are made as described above.
Daughter 96-well plates may be spotted (3.2 μL/well) from the stock plates listed above using the BioMek FX robot and used immediately or frozen at −70° C. until use. Aerobic organisms are inoculated (100 μL volumes) into the thawed plates using the BioMek FX robot. The inoculated plates are be placed in stacks and covered with an empty plate. These plates are then incubated for 16 to 24 hours in ambient atmosphere according to CLSI guidelines (National Committee for Clinical Laboratory Standards, Methods for Dilution, Antimicrobial Tests for Bacteria that Grow Aerobically; Approved Standard-Sixth Edition. NCCLS document M7-A6 {ISBN 1-56238-486-4}).
After inoculation and incubation, the degree of bacterial growth can be estimated visually with the aid of a Test Reading Mirror (Dynex Technologies 220 16) in a darkened room with a single light shining directly through the top of the microbroth tray. The MIC is the lowest concentration of drug that prevents macroscopically visible growth under the conditions of the test.
Additionally, any one or more of the saturated acyl guanidine compounds described herein can be used to treat a F1F0-ATP hydrolase associated disorder (e.g., myocardial infarction, ventricular hypertrophy, coronary artery disease, non-Q wave MI, congestive heart failure, cardiac arrhythmias, unstable angina, chronic stable angina, Prinzmetal's angina, high blood pressure, intermittent claudication, peripheral occlusive arterial disease, thrombotic or thromboembolic symptoms of thromboembolic stroke, venous thrombosis, arterial thrombosis, cerebral thrombosis, pulmonary embolism, cerebral embolism, thrombophilia, disseminated intravascular coagulation, restenosis, atrial fibrillation, ventricular enlargement, atherosclerotic vascular disease, atherosclerotic plaque rupture, atherosclerotic plaque formation, transplant atherosclerosis, vascular remodeling atherosclerosis, cancer, surgery, inflammation, systematic infection, artificial surfaces, interventional cardiology, immobility, medication, pregnancy and fetal loss, and diabetic complications comprising retinopathy, nephropathy and neuropathy) in a subject.
Combination Therapy
Additionally, the guanidine compounds described herein can be used in combination with at least one other therapeutic agent, such as potassium channel openers, calcium channel blockers, sodium hydrogen exchanger inhibitors, antiarrhythmic agents, antiatherosclerotic agents, anticoagulants, antithrombotic agents, prothrombolytic agents, fibrinogen antagonists, diuretics, antihypertensive agents, ATPase inhibitors, mineralocorticoid receptor antagonists, phospodiesterase inhibitors, antidiabetic agents, anti-inflammatory agents, antioxidants, angiogenesis modulators, antiosteoporosis agents, hormone replacement therapies, hormone receptor modulators, oral contraceptives, antiobesity agents, antidepressants, antianxiety agents, antipsychotic agents, antiproliferative agents, antitumor agents, antiulcer and gastroesophageal reflux disease agents, growth hormone agents and/or growth hormone secretagogues, thyroid mimetics, anti-infective agents, antiviral agents, antibacterial agents, antifungal agents, cholesterol/lipid lowering agents and lipid profile therapies, and agents that mimic ischemic preconditioning and/or myocardial stunning, antiatherosclerotic agents, anticoagulants, antithrombotic agents, antihypertensive agents, antidiabetic agents, and antihypertensive agents selected from ACE inhibitors, AT-1 receptor antagonists, ET receptor antagonists, dual ET/AII receptor antagonists, vasopepsidase inhibitors, an antiplatelet agent selected from GPIIb/IIIa blockers, P2Y1 and P2Y12 antagonists, thromboxane receptor antagonists, or aspirin, along with a pharmaceutically-acceptable carrier or diluent in a pharmaceutical composition.
IV. Pharmaceutical Compositions, Formulations, and Exemplary Administration Routes and Dosing Considerations
Exemplary embodiments of various contemplated medicaments and pharmaceutical compositions are provided below.
A. Preparing Medicaments
Compounds of the present invention are useful in the preparation of medicaments to treat a variety of conditions, such as conditions associated with dysregulation of cell death, aberrant cell growth and hyperproliferation. One of skill in the art will appreciate that any one or more of the compounds described herein, including the many specific embodiments, are prepared by applying standard pharmaceutical manufacturing procedures. Such medicaments can be delivered to the subject by using delivery methods that are well-known in the pharmaceutical arts.
B. Exemplary Pharmaceutical Compositions and Formulation
In some embodiments of the present invention, the compositions are administered alone, while in some other embodiments, the compositions are preferably present in a pharmaceutical formulation comprising at least one active ingredient/agent, as discussed above, together with a solid support or alternatively, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic agents (e.g., those described in section III hereinabove). Each carrier should be “acceptable” in the sense that it is compatible with the other ingredients of the formulation and not injurious to the subject.
Contemplated formulations include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration. In some embodiments, formulations are conveniently presented in unit dosage form and are prepared by any method known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association (e.g., mixing) the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, wherein each preferably contains a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. In other embodiments, the active ingredient is presented as a bolus, electuary, or paste, etc.
In some embodiments, tablets comprise at least one active ingredient and optionally one or more accessory agents/carriers are made by compressing or molding the respective agents. In some embodiments, compressed tablets are prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Molded tablets are made by molding in a suitable machine a mixture of the powdered compound (e.g., active ingredient) moistened with an inert liquid diluent. Tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Pharmaceutical compositions for topical administration according to the present invention are optionally formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. In alternative embodiments, topical formulations comprise patches or dressings such as a bandage or adhesive plasters impregnated with active ingredient(s), and optionally one or more excipients or diluents. In some embodiments, the topical formulations include a compound(s) that enhances absorption or penetration of the active agent(s) through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide (DMSO) and related analogues.
If desired, the aqueous phase of a cream base includes, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
In some embodiments, oily phase emulsions of this invention are constituted from known ingredients in a known manner. This phase typically comprises a lone emulsifier (otherwise known as an emulgent), it is also desirable in some embodiments for this phase to further comprise a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier so as to act as a stabilizer. In some embodiments it is also preferable to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.
The choice of suitable oils or fats for the formulation is based on achieving the desired properties (e.g., cosmetic properties), since the solubility of the active compound/agent in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus creams should preferably be non-greasy, non-staining and washable products with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the agent.
Formulations for rectal administration may be presented as a suppository with suitable base comprising, for example, cocoa butter or a salicylate.
Formulations suitable for vaginal administration may be presented as pessaries, creams, gels, pastes, foams or spray formulations containing in addition to the agent, such carriers as are known in the art to be appropriate.
Formulations suitable for nasal administration, wherein the carrier is a solid, include coarse powders having a particle size, for example, in the range of about 20 to about 500 microns which are administered in the manner in which snuff is taken, i.e., by rapid inhalation (e.g., forced) through the nasal passage from a container of the powder held close up to the nose. Other suitable formulations wherein the carrier is a liquid for administration include, but are not limited to, nasal sprays, drops, or aerosols by nebulizer, and include aqueous or oily solutions of the agents.
Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. In some embodiments, the formulations are presented/formulated in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, as herein above-recited, or an appropriate fraction thereof, of an agent.
It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents.
It also is intended that the agents, compositions and methods of this invention be combined with other suitable compositions and therapies. Still other formulations optionally include food additives (suitable sweeteners, flavorings, colorings, etc.), phytonutrients (e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, and other acceptable compositions (e.g., conjugated linoelic acid), extenders, and stabilizers, etc.
C. Exemplary Administration Routes and Dosing Considerations
Various delivery systems are known and can be used to administer therapeutic agents (e.g., exemplary compounds as described above) of the present invention, e.g., encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis, and the like. Methods of delivery include, but are not limited to, intra-arterial, intra-muscular, intravenous, intranasal, and oral routes. In specific embodiments, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, injection, or by means of a catheter.
The agents identified can be administered to subjects or individuals susceptible to or at risk of developing pathological growth of target cells and correlated conditions. When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject. To identify patients that can be beneficially treated, a tissue sample is removed from the patient and the cells are assayed for sensitivity to the agent.
Therapeutic amounts are empirically determined and vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the agent. When delivered to an animal, the method is useful to further confirm efficacy of the agent. One example of an animal model is MLR/MpJ-lpr/lpr (“MLR-lpr”) (available from Jackson Laboratories, Bar Harbor, Me.). MLR-lpr mice develop systemic autoimmune disease. Alternatively, other animal models can be developed by inducing tumor growth, for example, by subcutaneously inoculating nude mice with about 105 to about 109 hyperproliferative, cancer or target cells as defined herein. When the tumor is established, the compounds described herein are administered, for example, by subcutaneous injection around the tumor. Tumor measurements to determine reduction of tumor size are made in two dimensions using venier calipers twice a week. Other animal models may also be employed as appropriate. Such animal models for the above-described diseases and conditions are well-known in the art.
In some embodiments, in vivo administration is effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations are carried out with the dose level and pattern being selected by the treating physician.
Suitable dosage formulations and methods of administering the agents are readily determined by those of skill in the art. Preferably, the compounds are administered at about 0.01 mg/kg to about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100 mg/kg, even more preferably at about 0.5 mg/kg to about 50 mg/kg. When the compounds described herein are co-administered with another agent (e.g., as sensitizing agents), the effective amount may be less than when the agent is used alone.
The pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granulates or powders. In addition to an agent of the present invention, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.
More particularly, an agent of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including, but not limited to, oral, rectal, nasal, topical (including, but not limited to, transdermal, aerosol, buccal and sublingual), vaginal, parental (including, but not limited to, subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It is also appreciated that the preferred route varies with the condition and age of the recipient, and the disease being treated.
Ideally, the agent should be administered to achieve peak concentrations of the active compound at sites of disease. This may be achieved, for example, by the intravenous injection of the agent, optionally in saline, or by oral administration, for example, as a tablet, capsule or syrup containing the active ingredient.
Desirable blood levels of the agent may be maintained by a continuous infusion to provide a therapeutic amount of the active ingredient within disease tissue. The use of operative combinations is contemplated to provide therapeutic combinations requiring a lower total dosage of each component than may be required when each individual therapeutic compound or drug is used alone, thereby reducing adverse effects.
D. Exemplary Co-Administration Routes and Dosing Considerations
The invention also includes methods involving co-administration of the compounds described herein with one or more additional active agents. Indeed, it is a further aspect of this invention to provide methods for enhancing prior art therapies and/or pharmaceutical compositions by co-administering a compound of this invention. In co-administration procedures, the agents may be administered concurrently or sequentially. In one embodiment, the compounds described herein are administered prior to the other active agent(s). The pharmaceutical formulations and modes of administration may be any of those described above. In addition, the two or more co-administered chemical agents, biological agents or radiation may each be administered using different modes or different formulations.
The agent or agents to be co-administered depend on the type of condition being treated. For example, when the condition being treated is cancer, the additional agent can be a chemotherapeutic agent or radiation. When the condition being treated is an immune disorder, the additional agent can be an immunosuppressant or an anti-inflammatory agent. When the condition being treated is chronic inflammation, the additional agent can be an anti-inflammatory agent. The additional agents to be co-administered, such as anticancer, immunosuppressant, anti-inflammatory, can be any of the well-known agents in the art, including, but not limited to, those that are currently in clinical use. The determination of appropriate type and dosage of radiation treatment is also within the skill in the art or can be determined with relative ease.
Treatment of the various conditions associated with abnormal apoptosis is generally limited by the following two major factors: (1) the development of drug resistance and (2) the toxicity of known therapeutic agents. In certain cancers, for example, resistance to chemicals and radiation therapy has been shown to be associated with inhibition of apoptosis. Some therapeutic agents have deleterious side effects, including non-specific lymphotoxicity, renal and bone marrow toxicity.
The methods described herein address both these problems. Drug resistance, where increasing dosages are required to achieve therapeutic benefit, is overcome by co-administering the compounds described herein with the known agent.
EXAMPLES
The invention now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.
Example 1
Preparation of Saturated Acyl Guanidine Compounds
Described below is an exemplary general synthetic procedure for making saturated acyl guanidine compounds, along with an exemplary synthetic procedure for making the specific saturated acyl guanidine compound (Z)—N-((tert-butylamino)((5-(4-fluorophenyl)-1H-pyrazol-3-yl)amino)methylene)-3,3-dimethylbutanamide.
Part I
General Method for Making Saturated Acyl Guanidine Compounds




Saturated acyl guanidines can be prepared from an acid chloride, a first amine (A1-NH2), and a second amine (R3—NH2) using a three-step, 2-pot procedure, where variables R1, A1, and R3 are, for example, as defined in association with Formula I in the detailed description above. First, the requisite acid chloride is combined with a slight molar excess of potassium thiocyanate in a polar aprotic solvent (such as acetonitrile) at a temperature in the range of from about 0° C. to room temperature. The resulting mixture is stirred for a time period ranging from 15 minutes to 18 hours to provide a reaction mixture containing the acyl isothiocyanate synthetic intermediate compound. This reaction mixture may be used directly in the next reaction or filtered to remove the potassium chloride generated during this first step.
In a second step, the acyl isothiocyanate compound is combined with a second amine (R3—NH2) (which may be dissolved in a solvent) to provide a reaction mixture that is stirred for a time period ranging from 15 minutes to 2 hours to produce an acyl thiourea product. The acyl thiourea product may precipitate from the reaction mixture and be collected by filtration. Water may be added to the reaction mixture (typically in amount equal to the volume of organic solvent) to facilitate collection of the acyl thiourea product. The acyl thiourea product is dried in vacuo at approximately 50° C.
In a third step, the acyl thiourea product is combined with a coupling agent (such as N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC)) and a second amine to produce the saturated acyl guanidine. All starting materials for this reaction may be combined prior to heating the reaction mixture or the EDC may be added last, once the temperature of the reaction mixture has been raised. Alternatively, the second amine may be added last after combining the thiourea and the EDC. Once all the starting materials have been combined, the reaction mixture is generally stirred for a time period ranging from about 30 minutes to about 24 hours while the reaction mixture is heated to a temperature ranging from about 45° C. to about 70° C.
Starting materials can be obtained from commercial sources or readily prepared from commercially available materials. For example, the acid chloride compounds acetyl chloride, cyclopropanecarbonyl chloride, methoxyacetyl chloride, cyclobutanecarbonyl chloride, 1-methyl-1-cyclohexanecarboxylic acid chloride, isobutyryl chloride, 2-cyclohexylacetyl chloride, 2-phenylacetyl chloride, 1-methyl cyclopentanecarbonyl chloride, cyclohexane carbonyl chloride, and pivaloyl chloride are commercially available, as well as the following amino-indazoles:




Furthermore, compounds can be characterized by high performance liquid chromatography (HPLC), mass spectrometry (MS) and/or 1H nuclear magnetic resonance spectroscopy. HPLC methods that may be used to characterize the guanidine compounds are as follows: Method A conditions were Waters Symmetry C-18 column, 4.6×150 mm, 3.5 micron, 23° C., 1.0 mL/min, 1 min 25% MeCN in H2O (0.1% TFA), 10 min gradient of 25%-95% MeCN in H2O (0.1% TFA), 95% MeCN in H2O (0.1% TFA) for 5 min, and then equilibration to 25% MeCN in H2O (0.1% TFA) over 2.0 min; Method B conditions were Agilent Zorbax C-18 column, 4.6×50 mm, 1.8 micron, 23° C., 1.0 mL/min, 1 min 25% MeCN in H2O (0.1% TFA), 5 min gradient of 25%-95% MeCN in H2O (0.1% TFA), 1 min at 95% MeCN in H2O (0.1% TFA), and then equilibration to 25% MeCN in H2O (0.1% TFA) over 1.0 min; Method C conditions were Phenomenex Kinetex C18 (3.0 mm×50 mm), 2.6 micron, 58° C., 1.5 mL/min, 4 min gradient 5% MeCN (0.1% TFA) in H2O (0.1% TFA) to 100% MeCN (0.1% TFA), 100% MeCN (0.1% TFA) for 0.5 min, and then equilibration to 5% MeCN (0.1% TFA) in H2O (0.1% TFA) over 1.5 min; Method D conditions were Phenomenex Kinetex C18 (3.0 mm×50 mm), 2.6 micron, 40° C., 1.5 mL/min, 4 min gradient 5% MeCN (0.1% TFA) in H2O (0.1% TFA) to 100% MeCN (0.1% TFA), 100% MeCN (0.1% TFA) for 0.5 min, and then equilibration to 5% MeCN (0.1% TFA) in H2O (0.1% TFA) over 1.5 min; Method E conditions were Waters Symmetry C18 (4.6 mm×150 mm), 3.5 micron, 26° C., 2.0 mL/min, 1 min 25% MeCN (0.05% TFA) in H2O (0.05% TFA), 7 min gradient of 25%-95% MeCN (0.05% TFA) in H2O (0.05% TFA), 95% MeCN (0.05% TFA) in H2O (0.05% TFA) for 2 min, and then equilibration to 25% MeCN (0.05% TFA) in H2O (0.05% TFA) over 2.0 min; and Method F conditions were Shim-pack XR-ODS, 2.2 micron, 40° C., 1.0 mL/min, 10 min gradient 5% MeCN (0.1% TFA) in H2O (0.1% TFA) to 100% MeCN (0.1% TFA), 100% MeCN (0.1% TFA) for 1 min, and then equilibration to 5% MeCN (0.1% TFA) in H2O (0.1% TFA) over 1 min. The phrase “MeCN (0.05% TFA)” is art-recognized and refers to acetonitrile containing 0.05% wt/wt trifluoroacetic acid. The phrase “H2O (0.1% TFA)” is art-recognized and refers to water containing 0.1% wt/wt trifluoroacetic acid.

Part II
Exemplary Synthetic Procedure for Preparing (Z)—N-((tert-Butylamino)((5-(4-fluorophenyl)-1H-pyrazol-3-yl)amino)methylene)-3,3-dimethylbutanamide (1)
The title compound was prepared according to the procedures described below.
Step A: Preparation N-((5-(4-fluorophenyl)-1H-pyrazol-3-yl)carbamothioyl)-3,3-dimethylbutanamide




3,3-Dimethylbutanoyl chloride (500 mg, 3.71 mmol) was dissolved in acetonitrile (18 mL) and potassium thiocyanate (397 mg, 4.09 mmol) was added. The resulting slurry was stirred at room temperature for approximately 30 minutes, then 5-(4-fluorophenyl)-1H-pyrazol-3-amine (658 mg, 3.71 mmol) was added in one portion. The reaction mixture was stirred at room temperature for an additional 30 minutes and concentrated. The residue was washed with water, then hexanes resulting in a solid. The solid was collected by filtration and dried at 50° C. in vacuo to provide 978 mg (79% yield) of the title compound as a solid. 1H NMR (DMSO-d6) δ 13.2 (s, 1H), 13.0 (s, 1H), 10.4 (s, 1H), 7.78 (m, 2H), 7.35 (2, 1H), 7.25 (m, 2H), 1.15 (s, 2H), 1.00 (s, 9H).
Step B: Preparation of (Z)—N-((tert-Butylamino)((5-(4-fluorophenyl)-1H-pyrazol-3-yl)amino)methylene)-3,3-dimethylbutanamide (1)




N-((5-(4-Fluorophenyl)-1H-pyrazol-3-yl)carbamothioyl)-3,3-dimethylbutanamide (400 mg, 1.20 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) (252 mg, 1.32 mmol) were combined in tetrahydrofuran (THF) (12 mL), and tert-butylamine (96.2 g, 1.32 mmol) was added. The resulting reaction mixture was then heated to 50° C. for 30 min and then the reaction mixture was allowed to cool, and concentrated on silica gel. The crude product was purified by silica gel chromatography (9:1 hexanes/EtOAC to 3:7 hexanes/EtOAc) to afford the title compound (10 mg, 2.2% yield). MS: calc.=373; obs.=374 (M++1). HPLC (Method B): retention time was 4.97 minutes.
Example 2
Preparation of (E)-N-(((1H-Indazol-6-yl)amino)((3-chlorophenyl)amino)methylene)cyclohexanecarboxamide (2)
The title compound was prepared according to the procedures described below.
Step A: Preparation of N-((3-Chlorophenyl)carbamothioyl)cyclohexanecarboxamide




To cyclohexylcarbonyl chloride (1.00 g, 6.82 mmol) in THF (10 mL) at 0° C. was added potassium thiocyanate (795 mg, 8.18 mmol). After 10 minutes, the cooling bath was removed and the reaction was allowed to stir at room temperature for 1 hour. To the resulting suspension was added 3-chloroaniline (870 mg, 6.82 mmol), and the suspension was stirred at ambient temperature overnight. Next, water was added to the reaction mixture, and the resulting suspension was filtered and dried in vacuo to afford the title compound as a pale yellow solid. HPLC (Method A): retention time was 11.60 minutes.
Step B: Preparation of (E)-N-(((1H-Indazol-6-yl)amino)((3-chlorophenyl)amino)methylene)cyclohexanecarboxamide (2)




N-((3-Chlorophenyl)carbamothioyl)cyclohexanecarboxamide (100 mg, 0.337 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) (78.0 mg, 0.404 mmol) and 1H-indazol-6-amine (45.0 mg, 0.337 mmol) were combined in N,N-dimethylformamide (DMF) (1.5 mL). Then, the resulting reaction mixture was heated to 65° C. overnight. Because the reaction was not complete by HPLC, and the reaction temperature was raised to 75° C. Heating was continued overnight. Next, the reaction mixture was allowed to cool, diluted with ethyl acetate, washed with water, and the organic fraction was concentrated onto silica gel. The crude product was purified by silica gel chromatography (30-50% EtOAc/hexanes) to afford the title compound as a white solid. 1H NMR (DMSO-d6) δ 12.8 (s, 1H), 9.6 (m, 2H), 6.8-8.4 (m, 8H), 6.8 (m, 2H), 2.1 (m, 1H), 1.6 (m, 4H), 1.1 (M, 6H). MS: calc.=395.89; obs.=395.90. HPLC (Method A): retention time was 6.71 minutes.
Example 3
Preparation of N-(((1H-Indazol-6-yl)amino)((3-chloro-5-fluorophenyl)amino)methylene)-2-(4-chlorophenyl)acetamide (3)
The title compound was prepared according to the procedures described below.
Part I
Preparation of N-((3-Chloro-5-fluorophenyl)carbamothioyl)-2-(4-chlorophenyl)acetamide




To 4-chlorophenacyl chloride (1.0 g, 5.3 mmol) in anhydrous acetonitrile (15 mL) under a nitrogen atmosphere was added potassium thiocyanate (0.57 g, 5.8 mmol). The suspension was stirred at ambient temperature for 2.5 hours. Next, to the suspension was added 3-chloro-5-fluoroaniline (0.85 g, 5.8 mmol), and the resulting mixture was stirred at ambient temperature for 2 hours. Then, water (30 mL) was slowly added to the reaction mixture, and the resulting mixture was slurried for 20 minutes. The solids were collected by filtration and washed with water then dried in vacuo to yield the title compound (1.72 g, 91%).
Part II
Preparation of N-(((1H-Indazol-6-yl)amino)((3-chloro-5-fluorophenyl)amino)methylene)-2-(4-chlorophenyl)acetamide (3)




In a round bottomed flask was combined N-((3-chloro-5-fluorophenyl)carbamothioyl)-2-(4-chlorophenyl)acetamide (0.2 g, 0.56 mmol) and 1H-indazol-6-amine (82 mg, 0.62 mmol), followed by EDC (0.13 g, 0.67 mmol) in N,N-dimethylformamide (3 mL) and this mixture was heated to 65° C. for 5 hours. Then, the solution was cooled, diluted with ethyl acetate, washed with water, then brine, dried with sodium sulfate, filtered and concentrated in vacuo to provide a crude product, which was purified by column chromatography (eluting with a gradient of ethyl acetate in hexanes) to provide the title compound (51 mg, 20%). MS: calc.=456.30; obs.=455.84, 457.80 (in positive ion mode). HPLC (Method A): retention time was 8.05 minutes.
Example 4
Preparation of 4-Fluoro-7-methoxy-1H-indazol-3-amine




2,6-Difluoro-3-methoxy-benzonitrile (1 g, 5.9 mmol) was dissolved in ethanol (20 mL) and treated with hydrazine (0.21 g, 6.5 mmol) in a glass vial. The vial was capped and placed on a shaker then heated to 75° C. overnight. Then, the mixture was evaporated to dryness and the product was obtained as a 1:1 mixture of isomers.
Example 5
Preparation of 6-(Trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridin-3-amine




To a solution of [5-(trifluoromethyl)-2-pyridyl]hydrazine (3.895 g, 21.99 mmol) in methanol (100 mL) was added cyanogen bromide solid (5.82 g, 54.9 mmol) all at once. The reaction mixture was stirred at room temperature overnight. Then, the resulting solid was filtered off and rinsed with a small amount of 1:1 EtOAc:MeOH, giving the indazole as the hydrobromide salt. The resulting solid salt (5.07 g) was dissolved in 250 mL of MeOH and treated with MP-carbonate resin (17.49 g: Aldrich catalog number 540293, 2.5-3.5 mmol/g). This mixture was stirred overnight and then the resin was filtered off, rinsing with alternating methanol and methylene chloride. The solvents were removed under vacuum to give the title compound (3.71 g, 83.4% yield) as a yellow/brown solid. 1H NMR (400 MHz, DMSO-d6) δ 8.77 (m, 1H), 7.62 (m, 1H), 7.24 (m, 1H), 6.74 (br s, 2H).
Example 6
Compounds described in above Examples were tested for activity against F1F0-ATPase by measuring the ability of the compounds to inhibit ATP synthesis. In addition, the compounds were assessed for cytotoxicity in Ramos cells. Results of the biological activity tests are shown in Table 4 below. Inhibition of F1F0-ATPase activity in synthesizing ATP and cytotoxicity in Ramos cells were measured according to the procedures described in K. M. Johnson et al. Chemistry & Biology 2005, 12, 485-496. In the column titled ATP Syn IC50 in Table 4, the symbol “+” refers to an IC50>16 μm, “++” refers to an IC50 from 5 to 16 μm, and “+++” refers to an IC50 less than 5 μm. In the column titled Ramos Cell EC50 in Table 4, the symbol “+” refers to an EC50>16 μm, “++” refers to an EC50 from 5 to 16 μm, and “+++” refers to an EC50 less than 5 μm.









TABLE 4





Compound
ATP Syn
Ramos Cell


No.
IC50 (μM)
EC50 (μM)







1
+++
++


2
++
++


3
+++
++









INCORPORATION BY REFERENCE
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.
EQUIVALENTS
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

## Claims

1. A compound represented by Formula I:



including all stereoisomers, geometric isomers, and tautomers; or a pharmaceutically acceptable salt or solvate of any of the foregoing; wherein:
A1 is pyrazolyl optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of phenyl, alkyl, haloalkyl, cycloalkyl, halogen, hydroxyl, hydroxyalkyl, and C1-6alkoxy, wherein the phenyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, hydroxyalkyl, C1-6alkoxy, and cyano;
R1 is alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, alkylene-O-alkyl, alkylene-cycloalkyl, alkylene-heterocycloalkyl, aralkyl, 2,3-dihydro-1H-inden-1-yl, 2,3-dihydro-1H-inden-2-yl, 1,2,3,4-tetrahydronaphthalen-1-yl, or 1,2,3,4-tetrahydronaphthalen-2-yl, wherein the cycloalkyl, heterocycloalkyl, aralkyl, 2,3-dihydro-1H-inden-1-yl, 2,3-dihydro-1H-inden-2-yl, 1,2,3,4-tetrahydronaphthalen-1-yl, and 1,2,3,4-tetrahydronaphthalen-2-yl are optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, cycloalkyl, hydroxyl, alkoxyl, and cyano;
R2 and R4 each represent independently hydrogen or alkyl;
R3 is one of the following:
(i) alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkoxyl, —O—(C(R5)2)m-alkoxyl, —N(R6)(R7), heterocycloalkyl, —N(R6)C(O)-alkyl, —C(O)N(R6)(R7), —N(R6)C(O)N(R6)(R7), halogen, haloalkyl, and cyano; or
(ii) cycloalkyl, —(C(R5)2)m-cycloalkyl, aryl, or aralkyl, each of which are optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, hydroxyalkyl, alkoxy, —S-alkyl, and cyano;

R5 represents independently for each occurrence hydrogen, alkyl, or cycloalkyl; or two occurrences of R5 attached to the same carbon atom are taken together with said carbon atom to form a saturated carbocylic ring;
R6 and R7 each represent independently for each occurrence hydrogen, alkyl, or cycloalkyl; or R6 and R7 are taken together with the nitrogen atom to which they are attached to form a 3 to 7 membered heterocyclic ring optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, and C1-6alkoxy; and
m is 1, 2, 3, 4, or 5.

2. The compound of claim 1, wherein A1 is pyrazolyl optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of alkyl, haloalkyl, cycloalkyl, halogen, and hydroxyl.

3. The compound of claim 1, wherein A1 is pyrazolyl substituted with haloalkyl, or A1 is pyrazolyl substituted with phenyl, wherein the phenyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen and haloalkyl.

4. The compound of claim 1, wherein A1 is pyrazolyl substituted with 1 or 2 substituents independently selected from the group consisting of alkyl and haloalkyl.

5. The compound of claim 1, wherein A1 is represented by:




wherein R* is haloalkyl.

6. The compound of claim 1, wherein A1 is one of the following:

7. A compound represented by Formula II:



including all stereoisomers, geometric isomers, and tautomers; or a pharmaceutically acceptable salt or solvate of any of the foregoing; wherein:
A1 is





wherein B2 is a 6-membered heteroaromatic group;
R1 is alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, alkylene-O-alkyl, alkylene-cycloalkyl, alkylene-heterocycloalkyl, aralkyl, 2,3-dihydro-1H-inden-1-yl, 2,3-dihydro-1H-inden-2-yl, 1,2,3,4-tetrahydronaphthalen-1-yl, or 1,2,3,4-tetrahydronaphthalen-2-yl; wherein the cycloalkyl, heterocycloalkyl, aralkyl, 2,3-dihydro-1H-inden-1-yl, 2,3-dihydro-1H-inden-2-yl, 1,2,3,4-tetrahydronaphthalen-1-yl, and 1,2,3,4-tetrahydronaphthalen-2-yl are optionally substituted by 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, cycloalkyl, hydroxyl, alkoxyl, and cyano;
R2 and R4 each represent independently hydrogen or alkyl;
R3 is one of the following:
(i) alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkoxyl, —O—(C(R5)2)m-alkoxyl, —N(R6)(R7), heterocycloalkyl, —N(R6)C(O)-alkyl, —C(O)N(R6)(R7), —N(R6)C(O)N(R6)(R7), halogen, haloalkyl, and cyano; or
(ii) cycloalkyl, —(C(R5)2)m-cycloalkyl, aryl, or aralkyl, each of which are optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, hydroxyalkyl, alkoxy, —S-alkyl, and cyano;

R5 represents independently for each occurrence hydrogen, alkyl, or cycloalkyl; or two occurrences of R5 attached to the same carbon atom are taken together with said carbon atom to form a saturated carbocylic ring;
R6 and R7 each represent independently for each occurrence hydrogen, alkyl, or cycloalkyl; or R6 and R7 are taken together with the nitrogen atom to which they are attached to form a 3 to 7 membered heterocyclic ring optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, and C1-6alkoxy;
R8 is hydrogen, or R8 is alkyl substituted by hydroxyl, —N(H)(R6), heterocycloalkyl, —N(H)C(O)R10, —C(O)N(H)(R6), or —N(H)C(O)N(R6)(R7);
R9 represents independently for each occurrence halogen, haloalkyl, alkyl, cycloalkyl, heterocycloalkyl, hydroxyl, C1-6alkoxy, or cyano;
R10 is alkyl, cycloalkyl, aryl, or aralkyl;
m is 1, 2, 3, 4, or 5; and
n is 0, 1, 2, 3, or 4.

8. The compound of claim 1, wherein R1 is alkyl.

9. The compound of claim 1, wherein R1 is cycloalkyl or alkylene-cycloalkyl, each of which is optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, hydroxyl, and alkoxyl.

10. The compound of claim 1, wherein R1 is aralkyl optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, haloalkyl, alkyl, cycloalkyl, hydroxyl, alkoxyl, and cyano.

11. The compound of claim 1, wherein R1 is benzyl optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, and alkoxyl.

12. The compound of claim 1, wherein R2 and R4 are hydrogen.

13. The compound of claim 1, wherein R3 is alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkoxyl, —O—(C(R5)2)m-alkoxyl, —N(R6)(R7), heterocycloalkyl, —N(R6)C(O)-alkyl, —C(O)N(R6)(R7), —N(R6)C(O)N(R6)(R7), halogen, haloalkyl, and cyano.

14. The compound of claim 1, wherein R3 is alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl and alkoxyl.

15. The compound of claim 1, wherein R3 is alkyl or cycloalkyl.

16. The compound of claim 1, wherein R3 is phenyl, benzyl, or —(C(R5)2)m-cycloalkyl, each of which are optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, hydroxyalkyl, alkoxy, —S-alkyl, and cyano.

17. The compound of claim 1, wherein R3 is phenyl substituted with 1 or 2 substituents independently selected from the group consisting of chloro, fluoro, and trifluoromethyl.

18. The compound of claim 1, wherein R5 is hydrogen, and R6 and R7 are hydrogen.

19. The compound of claim 1, wherein said compound is represented by Formula I-A1:



including all stereoisomers, geometric isomers, and tautomers; or a pharmaceutically acceptable salt or solvate of any of the foregoing; wherein:
R1 is C1-6 alkyl, C1-6 haloalkyl, C3-6 cycloalkyl, C1-6 alkylene-O—C1-6 alkyl, C1-6 alkylene- C3-6 cycloalkyl, or C1-6 alkylene-heterocycloalkyl, wherein the cycloalkyl and heterocycloalkyl are optionally substituted by 1 or 2 substituents independently selected from the group consisting of halogen, haloalkyl, and alkyl;
R3 is one of the following:
(i) C1-6 alkyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl and alkoxyl; or
(ii) C3-6 cycloalkyl or —(CH2)m-cycloalkyl, each of which is optionally substituted with 1 or 2 substituents independently selected from the group consisting of hydroxyl, alkyl, hydroxyalkyl, and alkoxy;

R* is phenyl, alkyl, haloalkyl, or cycloalkyl; wherein the phenyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, and C1-6alkoxy; and
m is 1, 2, or 3.

20. The compound of claim 19, wherein R1 is C1-6 alkyl.

21. The compound of claim 19, wherein R1 is C3-6 cycloalkyl or C1-6 alkylene-C3-6 cycloalkyl, wherein the cycloalkyl is optionally substituted by 1 or 2 substituents independently selected from the group consisting of halogen, haloalkyl, and alkyl.

22. The compound of claim 19, wherein R3 is C1-6 alkyl optionally substituted by alkoxyl.

23. The compound of claim 19, wherein R* is haloalkyl.

24. The compound of claim 19, wherein R* is trifluoromethyl.

25. A compound listed in any one of Tables 1-4 or a pharmaceutically acceptable salt thereof:










TABLE 1







No.
Compound









I-1 







 



I-2 







 



I-3 







 



I-4 







 



I-5 







 



I-6 







 



I-7 







 



I-8 







 



I-9 







 



I-10







 



I-11







 



I-12







 



I-13







 



I-14







 



I-15







 



I-16







 



I-17







 



I-18







 



I-19







 



I-20







 



I-21







 



I-22







 



I-23







 



I-24







 



I-25







 



I-26







 



I-27







 



I-28







 



I-29







 



I-30







 



I-31







 



I-32







 



I-33







 



I-34







 



I-35







 



I-36







 



I-37







 



I-38







 



I-39







 



I-40





















TABLE 2













 










No.
A
X
Y





II-1 








isobutyl


 


II-2 








sec-butyl


 


II-3 








tert-butyl


 


II-4 








ethyl


 


II-5 








propyl


 


II-6 








isopropyl


 


II-7 








—C(H)(Me)CH2OMe


 


II-8 








—CH2CH2OMe


 


II-9 








—CH2CH2OEt


 


II-10








cyclopropyl


 


II-11








cyclopentyl


 


II-12








cyclohexyl


 


II-13








1-methylcyclobutyl


 


II-14








1-methylcyclopentyl


 


II-15








1-methylcyclohexyl


 


II-16
—CF3




isobutyl


 


II-17
—CF3




sec-butyl


 


II-18
—CF3




tert-butyl


 


II-19
—CF3




ethyl


 


II-20
—CF3




propyl


 


II-21
—CF3




isopropyl


 


II-22
—CF3




—C(H)(Me)CH2OMe


 


II-23
—CF3




—CH2CH2OMe


 


II-24








—CH2CH2OEt


 


II-25








cyclopropyl


 


II-26








cyclopentyl


 


II-27








cyclohexyl


 


II-28








1-methylcyclobutyl


 


II-29








1-methylcyclopentyl


 


II-30








1-methylcyclohexyl


 


II-31








isobutyl


 


II-32








sec-butyl


 


II-33








isopropyl


 


II-34








—C(H)(Me)CH2OMe


 


II-35








—C(H)(Me)CH2OMe


 


II-36








isobutyl


 


II-37








sec-butyl


 


II-38








tert-butyl


 


II-39








isopropyl


 


II-40








—C(H)(Me)CH2OMe


 


II-41














 


II-42














 


II-43
—CF3










 


II-44














 


II-45































TABLE 3







No.
Compound









III-1 







 



III-2 







 



III-3 







 



III-4 







 



III-5 







 



III-6 







 



III-7 







 



III-8 







 



III-9 







 



III-10







 



III-11







 



III-12







 



III-13







 



III-14







 



III-15







 



III-16







 



III-17







 



III-18







 



III-19







 



III-20







 



III-21







 



III-22







 



III-23







 



III-24







 



III-25







 



III-26







 



III-27







 



III-28







 



III-29







 



III-30







 



III-31







 



III-32







 



III-33







 



III-34







 



III-35







 



III-36





















TABLE 4













 










No.
A
X
Y





IV-1 








isobutyl


 


IV-2 








sec-butyl


 


IV-3 








tert-butyl


 


IV-4 








ethyl


 


IV-5 








propyl


 


IV-6 








isopropyl


 


IV-7 








—C(H)(Me)CH2OMe


 


IV-8 








—CH2CH2OMe


 


IV-9 








—CH2CH2OEt


 


IV-10








cyclopropyl


 


IV-11








cyclopentyl


 


IV-12








cyclohexyl


 


IV-13








1-methylcyclobutyl


 


IV-14








1-methylcyclopentyl


 


IV-15








4-methylcyclohexyl


 


IV-16








isobutyl


 


IV-17








sec-butyl


 


IV-18








tert-butyl


 


IV-19








ethyl


 


IV-20








propyl


 


IV-21








isopropyl


 


IV-22
—CF3




—C(H)(Me)CH2OMe


 


IV-23
—CF3




—CH2CH2OMe


 


IV-24
—CF3




—CH2CH2OEt


 


IV-25
—CF3




cyclopropyl


 


IV-26
—CF3




cyclopentyl


 


IV-27
—CF3




cyclohexyl


 


IV-28
—CF3




1-methylcyclobutyl


 


IV-29








1-methylcyclopentyl


 


IV-30








4-methylcyclohexyl


 


IV-31








isobutyl


 


IV-32








sec-butyl


 


IV-33








isopropyl


 


IV-34








—C(H)(Me)CH2OMe


 


IV-35








—C(H)(Me)CH2OMe


 


IV-36








isobutyl


 


IV-37








sec-butyl


 


IV-38








tert-butyl


 


IV-39








isopropyl


 


IV-40








—C(H)(Me)CH2OMe


 


IV-41














 


IV-42














 


IV-43














 


IV-44














 


IV-45














 


IV-46








isobutyl


 


IV-47








sec-butyl


 


IV-48








isopropyl


 


IV-49








—C(H)(Me)CH2OMe


 


IV-50








—C(H)(Me)CH2OMe


 


IV-51








isobutyl


 


IV-52








1-methylcyclopentyl


 


IV-53
—CCF3




cyclohexyl


 


IV-54














 


IV-55

26. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.

27. A method of treating a disorder associated with dysregulation of cell death selected from the group consisting of an immune disorder, inflammatory disorder, cardiovascular disease, myeloma, lymphoma, cancer, and bacterial infection, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of claim 1 in order to ameliorate a symptom of the disorder.

28. The method of claim 27, wherein the disorder is rheumatoid arthritis, psoriasis, chronic graft-versus-host disease, acute graft-versus-host disease, Crohn's disease, inflammatory bowel disease, multiple sclerosis, systemic lupus erythematosus, Celiac Sprue, idiopathic thrombocytopenic thrombotic purpura, myasthenia gravis, Sjogren's syndrome, scleroderma, ulcerative colitis, asthma, uveitis, or epidermal hyperplasia.

29. The method of claim 27, wherein the patient is a human.

30. A method of inhibiting a F1F0-ATPase, comprising exposing a F1F0-ATPase to a compound of claim 1 to inhibit said F1F0-ATPase.