WO2023158845A1 - Small molecules targeting the vdac nadh-binding pocket to modulate cancer metabolism - Google Patents

Abstract

The present invention relates to VD AC-targeting small molecules that induce mitochondrial dysfunction and inhibit cell proliferation for the treatment of, for example, cancer.

Description

SMALL MOLECULES TARGETING THE VDAC NADH-BINDING POCKET TO MODULATE CANCER METABOLISM

FIELD OF THE INVENTION

The invention relates generally to small molecule pharmaceutical compounds that target the VDAC NADH-binding pocket and are useful for the treatment of cancer. All documents cited to or relied upon below are expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under U.S. National Institutes of Health (NIH/NCI) RO1 CA184456 and ULI TR001450-SCTR 2011. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Cancer bioenergetics and cancer metabolism are contributed both by aerobic glycolysis and oxidative phosphorylation (Oxphos). In the early 20th century, Warburg showed that at physiological partial pressures of oxygen, tumors produce more lactic acid than non-tumor tissues (1, 2). Warburg even proposed that damaged mitochondria were the origin of cancer, a theory proved wrong shortly after. In fact, both high glycolysis and oxidative metabolism contribute to tumor cell metabolism in different proportions (3-8). The current consensus is that the Warburg phenotype favors proliferation by providing carbon backbones and metabolic intermediaries needed for the synthesis of biomass (9-11). The relative contribution of mitochondria to generation of ATP and metabolic intermediaries, varies among tumor types, within the same tumor, and during tumor growth, indicating a spatiotemporal dynamic regulation of oxidative metabolism. The interdependency between enhanced glycolysis and mitochondrial metabolism, is a key feature contributing to the metabolic heterogeneity, increasingly recognized as a cause of failure in chemotherapy. Thus, mitochondria appear as an attractive target to develop novel “metabolic” treatments to inhibit cell proliferation (12-14).

Maintenance of mitochondrial metabolism depends on an adequate influx of respiratory substrates, cytosolic ATP, ADP, Pi and small ions into mitochondria; and the efflux of ATP and metabolic intermediaries to the cytosol. To reach the mitochondrial matrix, most anionic metabolites and small ions cross the outer mitochondrial membrane (OMM) through Voltage dependent anion channels (VDAC). VDAC (~30 kDa), is the most abundant protein in the outer mitochondrial membrane, comprising three isoforms. In most mammalian cells, VDAC1 and 2 are the most abundant, whereas VDAC3 is the least expressed isoform; except for testis and spermatozoa (15, 16). NMR and X-ray crystallography have shown that VDAC1 and 2, form a transmembrane P-barrel protein, with 19 anti-parallel P-strands and an N-terminal a-helical region located within the pore (17, 18). VDAC inserted in non-polarized or weakly polarized membranes (close to 0 mV), stays mostly in the high conductance open state. By contrast, applied positive or negative membrane potentials induce conformational changes to several lower conductance, closed states (maximal at -45 mV or +45 mV) (19, 20). Although gating and selectivity for VDAC1 and VDAC2 are very similar in different cell types, the detailed molecular determinants of voltage gating are still incompletely understood.

Regardless of the mechanism controlling gating, the flux of polar metabolites through VDAC is determined mostly by their charge and size (Colombini, 1980; Colombini, 2004). VDAC is selective for anionic metabolites and small cations (21-23). The open state of VDAC allows the flux of anions including most respiratory substrates, ATP4-, ADP3-, AMP, HPO42-, and phosphocreatine, among others. In the closed states, VDAC favors a non-selective flux of cations including Na+, K+, and Ca2+ (24-26). Consequently, physiological or pharmacological regulations that induce a change from the open state to the closed states of VDAC, decrease and increase the flux of negatively charged metabolic substrates and cations, respectively. Even though VDAC was initially considered to be constitutively an “all-time open gateway,” allowing the unrestricted flux of metabolites, subsequent research showed multiple endogenous regulations. NADH, a/p tubulin heterodimers, hexokinase, alpha-synuclein, p53, bcl2 family members and mitochondrial creatine kinase among others regulate VDAC opening (27-29); (30, 31); (32). Post-translational modifications, mainly phosphorylation by protein kinases, GSK3P, PKA, and protein kinase C epsilon (PKCs), inhibit association of VDAC with other proteins and also regulates VDAC opening (33-35).

Overall, the movement of metabolites through VDAC depends on the concentration gradient across the OMM, the electric field, the number of channels, the selectivity-permeability to a particular metabolite, and the open probability of the channel. Thus, VDAC emerges as a potential pharmacological target to modulate mitochondrial metabolism in cancer cells. However, major limitations to develop VDAC -targeting drugs have been the isoform specificity, the lack of an identified druggable region, and a poor understanding of mechanisms regulating gating of the channel. Moreover, the mechanisms of action of several compounds, claimed to bind to VDAC, remain unknown. Recently, an NADH-binding pocket in VDAC1, with an amino acid sequence conserved in all VDAC isoforms, has been identified (36). The P-NADH binding to the pocket reduces VDAC conductance by steric occlusion of the pore without changes at the a-helix, suggesting that molecules binding to this site may prevent VDAC closing by endogenous regulators.

A need exists in the art for VDAC -targeting drugs as novel cancer treatments to inhibit cell proliferation by modulating mitochondrial metabolism.

SUMMARY OF THE INVENTION

The present invention is directed to compounds of formula I:

and pharmaceutically acceptable salts thereof.

The present invention is also directed to a pharmaceutical composition, comprising a therapeutically effective amount of a compound according to formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The present invention is further directed to a method for inducing mitochondrial dysfunction, a method for inhibiting cell proliferation, and a method for the treatment of cancer, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a drug screening strategy. In silico screening of the South Carolina Compound Collection SC³ was used for a shape-based query for compounds structurally similar to NADH, or to the docking surface of the NADH binding pocket on VDAC. In silico selected compounds were used in a cell based, phenotypic screening, to identify compounds based on the capability of causing a minimum of 40% change in A m. After identification of SC18, as the most potent compound, other bioenergetics parameters were evaluated. Figure 2 shows that SC-18 binds to VDAC. A) Structure of SCI 8 B) SC18 decreased the inflection temperature at which the protein unfolds, similar to NADH binding. C) Docking of SC18 overlaid with the native docking site of NADH of VDAC 1, revealed pose depicting points of interaction similar to NADH. Note the quinazoline-dione scaffold that forms two hydrogen bonds with Lys20, one with the backbone and with the terminal nitrogen, the other being a similar interaction to the ring oxygen from the sugar ring closest to the 1, 4-dihydronicotinamide moiety of NADH. Two pi-cation interactions were found between the quinazoline-dione and Arg218. The 6-nitro substitution formed a hydrogen bond with Tyr22 similar to the phosphate backbone of NADH. Finally, the trifluoromethyl overlaid with the amide moiety of the 1, 4-dihydronicotinamide of NADH in the binding pocket.

Figure 3 shows that C18 decreases mitochondrial membrane potential. HepG2 cells treated with SC18 (25 pM) for 2 h were loaded with TMRM, as described in Materials and Methods. A) SC18 decreased A m in WT, VDAC 1/2 KO, 2/3 KO, and 1/3 KO cells. Representative images pseudocoloured according to the reference bar. B) Quantification of TMRM relative fluorescence after SC18; C) The half maximal effective concentration of SC18 (EC50) was determined using TMRM fluorescence as a readout. * p<0.05 from 3 independent experiments. 4-5 randomly selected fields with 10-20 cells/field were analyzed, a.u.: arbitrary units.

Figure 4 shows short-term treatment with SC 18 modulates cellular bioenergetics. Cells were treated with SC18 for 2 h. A) SC18 decreased mitochondrial NADH, assessed by multiphoton confocal microscopy, as described in Materials and Methods. NADH autofluorescence were converted to gray scale; B) Quantitative analysis of NADH autofluorescence; C) Basal respiration after SC-18 was determined using a Seahorse XFe96 Analyzer, as described in Material & Methods. D) Cellular ATP was measured using a luciferase based assay. *p<0.05 from 3 independent experiments. 4-5 randomly selected fields with 10-20 cells/field were analyzed for NADH autofluorescence, a.u.: arbitrary units Figure 5 shows that SC18 decreased cell proliferation. A) Half the lethal dose of SC18 (LC50: 23 pM) was calculated from trypan blue exclusion assays; B-C) Wound healing assay: cells were treated with SC18 (25 pM), SOR (5 pM), or vehicle for 48 h. Wound healing was measured at 8 h intervals. Quantitative analysis (B); representative images (C). *p<0.05 from 3 independent experiments.

Figure 6 shows the synthesis protocol of representative compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for the purpose of clarity, many other elements found in typical pharmaceutical compositions. Those of ordinary skill in the art will recognize that other elements and/or steps are desirable and/or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein is directed to all such variations and modifications to such elements and methods known to those skilled in the art. Furthermore, the embodiments identified and illustrated herein are for exemplary purposes only, and are not meant to be exclusive or limited in their description of the present invention.

The invention is directed to small molecules targeting the NADH-binding pocket of VDAC that block the binding of NADH and possibly other endogenous regulators to the site, maintaining the channel in an open configuration. Using an in silico approach followed by biological assays, the inventors identified the lead compound SC18. Short-term treatment with SC18, decreased mitochondrial membrane potential (ATm), NADH and ATP, and increased basal respiration. Long-term exposure to SC 18 decreased cell proliferation and promoted cell death in a dosedependent fashion. Results suggests that decreased cell proliferation and cell death induced by SC 18 is mediated by mitochondrial dysfunction.

In almost all eukaryotic proliferating and non-proliferating cells, VDAC1 and 2 are the most abundant isoforms, except for testis and spermatozoa in which VDAC3 is more abundant. Regardless of the extensive research on VDAC structure and function in reconstituted systems, many questions about VDAC regulation and function in physiological and pathological conditions remain undetermined. A potential redundancy in function, and different metabolic roles for each isoform, proposed as likely happening in the intracellular milieu, has not been supported by experimental data. From a pharmacological perspective, the success of modulating VDAC opening is, most likely to occur if the three isoforms are targeted simultaneously.

Overall, VDAC opening and closing states are major determinants of mitochondrial metabolism (42-44, 45). However, VDAC isoform specificity and lack of identifiable druggable sites, have been big obstacles for the development of drugs regulating VDAC opening {Reina, 2017 #2444).

In previous work, the inventors showed that VDAC1, 2 and 3 are major contributors to mitochondrial metabolism in cancer cells (27). Single or double kd of VDAC isoforms in all combinations, decreased AT. In particular, kd of the minor isoform VDAC3 in HepG2 cells caused the maximum decrease in ATm, ATP and NADH {Maldonado, 2013 #1649, 46). The inventors also demonstrated that free tubulin dynamically modulates ATm and inhibits VDAC1 and VDAC2 in cancer cells (4, 27). Additional work from the inventors identified erastin and a series of erastin-like molecules as antagonists of the VDAC -tubulin interaction (47). VDAC opening induced by VDAC -tubulin antagonists led to increased oxidative metabolism, accumulation of ROS, mitochondrial dysfunction, reversal of the Warburg metabolism and cell death (46, 47).

Recently, it has been shown that binding of NADH to a specific pocket in VDAC1 induces closure of the channel. The inventors analyzed the sequence of residues in the VDAC1 NADH- binding pocket and found that it is fully conserved in VDAC2 and 3. Thus, the inventors hypothesized that the NADH-binding pocket was an excellent pharmacological target to identify small molecules to occupy the site, prevent the binding of endogenous regulators, and maintain all VDAC isoforms open. Because of the stereochemical characteristics of the pocket, the inventors used the nicotinamide moiety and adjacent phosphate groups to generate shape-based queries to screen in silico the South Carolina Compound Collection SC3. After subsequent modeling including molecular docking of initial candidates, the inventors identified SC 18 as a lead compound. The inventors also proved that SC 18 binds to VDAC1. The thermal shift assay that the inventors used indicates if the compound binds or not to the protein but has the limitation of not identifying specifically an intramolecular binding site. A precise determination of SC 18 binding to the NADH-binding pocket would require soaking VDAC1 crystals in the presence of the compound followed by NMR to identify the binding.

The in silico strategy of the inventors was followed by biological assays to identify the most potent lead. Initially, the inventors used ATm as the readout because it is a global indicator of mitochondrial function. Maintenance of ATm depends on an adequate flux of metabolites through VDAC, but also on the activity of the Krebs cycle and coupled oxidative phosphorylation. However, ATm is subjected to multiple levels of regulation and increases or decreases in AT do not necessarily imply a change in VDAC opening or closing states. Because of this caveat and to use a multidimensional approach, the inventors studied other indicators of mitochondrial function.

After identifying SC 18 as the most potent compound decreasing ATm, the inventors studied mitochondrial NADH generation, ATP production and respiration. Most mitochondrial NADH is produced by the oxidation of respiratory substrates in the Krebs cycle. NADH, in turn, is the main electron donor for the respiratory chain. Electrons originated from NADH enters the chain at complex I, and flow through complexes II, III, and IV to the final acceptor oxygen. The activity of the ETC generates protons pumped to the intermembrane space. Those protons are the main component of the proton motive force that maintains AT and is used to generate ATP by the ATP synthase. Similar to A m, NADH and ATP generation decreased in the presence of SC18. Although decreased A m, NADH and ATP would be consistent with decreased mitochondrial metabolism, the increase in respiration induced by the lead compound, seems to contradict this interpretation. It is possible that SC 18 exerts a time dependent biphasic effect. If SC 18 blocks the access of endogenous regulators to the NADH-binding pocket, it would be reasonable to expect a transient increase in mitochondrial metabolism followed by mitochondrial dysfunction. Thus, decreased A m, ATP, and NADH would reflect mitochondrial dysfunction. The increase in respiration induced by SC 18 is intriguing. Plausible explanations could be a direct mild uncoupling effect, independent of VDAC opening, or a down-regulation of pyruvate dehydrogenase kinase that will leave more pyruvate available for oxidation, among other mechanisms. In any case, more time-course studies will be necessary to add mechanistic explanations to the effects of SC 18. Overall, the findings of the inventors indicate that SC 18 decreases mitochondrial metabolism.

Mitochondria, that contribute 10-90% of total cellular ATP in cancer cells, is also an essential source of metabolic intermediaries for the synthesis of biomass. Thus, changes in mitochondrial metabolism may impact very differently not only cellular metabolism but also cell proliferation. In that sense, the inventors evaluated if the decrease in mitochondrial function was followed by a decrease in cell proliferation. The inventors showed that long-term treatment with SC 18 (3-5 d), delays wound healing. Since wound healing assays assess not only proliferation but migration as well, the inventors confirmed the inhibitory effect of SC 18 on cell proliferation by quantifying cell viability and proliferation using Trypan blue exclusion and direct counting. The results clearly indicated an inhibitory effect of SC 18 on cell proliferation starting at 10 pM. However, cell death was only observed (-96%) at maximal concentrations (30 pM).

A compound according to the invention is inherently intended to comprise all stereochemically isomeric forms thereof. The term "stereochemically isomeric forms" as used hereinbefore or hereinafter defines all the possible stereoisomeric forms which the compounds of formula (I) and their N-oxides, pharmaceutically acceptable salts or physiologically functional derivatives may possess. Unless otherwise mentioned or indicated, the chemical designation of compounds denotes the mixture of all possible stereochemically isomeric forms. In particular, stereogenic centers may have the R- or S-configuration; substituents on bivalent cyclic (partially) saturated radicals may have either the cis- or trans-configuration. Compounds encompassing double bonds can have an E (entgegen) or Z (zusammen)-stereochemistry at said double bond. The terms cis, trans, R, S, E and Z are well known to a person skilled in the art.

Stereochemically isomeric forms of the compounds of formula (I) are obviously intended to be embraced within the scope of this invention. Of special interest are those compounds of formula (I) which are stereochemically pure.

Following CAS-nomenclature conventions, when two stereogenic centers of known absolute configuration are present in a molecule, an R or S descriptor is assigned (based on Cahn-Ingold- Prelog sequence rule) to the lowest-numbered chiral center, the reference center. The configuration of the second stereogenic center is indicated using relative descriptors [R*,R*] or [R*,S*], where R* is always specified as the reference center and [R*,R*] indicates centers with the same chirality and [R*,S*] indicates centers of unlike chirality. For example, if the lowest- numbered chiral center in the molecule has an S configuration and the second center is R, the stereo descriptor would be specified as S— [R*,S*]. If "a" and " " are used: the position of the highest priority substituent on the asymmetric carbon atom in the ring system having the lowest ring number, is arbitrarily always in the "a" position of the mean plane determined by the ring system. The position of the highest priority substituent on the other asymmetric carbon atom in the ring system relative to the position of the highest priority substituent on the reference atom is denominated "a", if it is on the same side of the mean plane determined by the ring system, or "0", if it is on the other side of the mean plane determined by the ring system.

When a specific stereoisomeric form is indicated, this means that said form is substantially free, i.e. associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, further preferably less than 2% and most preferably less than 1% of the other isomer(s). Thus, when a compound of formula (I) is for instance specified as (R,S), this means that the compound is substantially free of the (S,R) isomer.

The compounds of formula (I) may be synthesized in the form of mixtures, in particular racemic mixtures, of enantiomers which can be separated from one another following art-known resolution procedures. The racemic compounds of formula (I) may be converted into the corresponding diastereomeric salt forms by reaction with a suitable chiral acid. Said diastereomeric salt forms are subsequently separated, for example, by selective or fractional crystallization and the enantiomers are liberated therefrom by alkali. An alternative manner of separating the enantiomeric forms of the compounds of formula (I) involves liquid chromatography using a chiral stationary phase. Said pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically. Preferably if a specific stereoisomer is desired, said compound will be synthesized by stereospecific methods of preparation. These methods will advantageously employ enantiomerically pure starting materials.

The tautomeric forms of the compounds of formula (I) are meant to comprise those compounds of formula (I) wherein e.g. an enol group is converted into a keto group (keto-enol tautomerism). Tautomeric forms of the compounds of formula (I) or of intermediates of the present invention are intended to be embraced by the ambit of this invention.

The term “alkyl” as used herein denotes an unbranched or branched chain, saturated, monovalent hydrocarbon residue containing 1 to 20 carbon atoms. In one embodiment, the number of carbon atoms in the alkyl chain can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. In another embodiment, the number of carbon atoms in the alkyl chain can be from 5 to 16 and referred to as "(C5-C16) alkyl.”

The term “lower alkyl” denotes a straight or branched chain hydrocarbon residue containing 1 to 6 carbon atoms. "C1-20 alkyl" as used herein refers to an alkyl composed of 1 to 20 carbons. Examples of alkyl groups include, but are not limited to, lower alkyl groups include methyl, ethyl, propyl, z-propyl, //-butyl, z-butyl, Lbutyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecycl and hexadecyl.

The term "alkenyl" as used herein denotes an unbranched or branched chain, saturated, monovalent hydrocarbon residue of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. In one embodiment, the number of carbon atoms in the alkenyl chain can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. In another embodiment, the number of carbon atoms in the alkenyl chain can be from 5 to 16 and referred to as "(Cs-Cie) alkenyl.”

When the term “alkyl” is used as a suffix following another term, as in “phenylalkyl,” or “hydroxyalkyl,” this is intended to refer to an alkyl group, as defined above, being substituted with one to two substituents selected from the other specifically-named group. Thus, for example, “phenylalkyl” denotes the radical R'R"-, wherein R' is a phenyl radical, and R" is an alkylene radical as defined herein with the understanding that the attachment point of the phenylalkyl moiety will be on the alkylene radical. Examples of arylalkyl radicals include, but are not limited to, benzyl, phenylethyl, 3 -phenylpropyl. The terms “arylalkyl” or "aralkyl" are interpreted similarly except R' is an aryl radical.

The term "alkoxy" as used herein means an -O-alkyl group, wherein alkyl is as defined above such as methoxy, ethoxy, //-propyloxy, z-propyloxy, //-butyl oxy, z-butyloxy, /-butyloxy, pentyloxy, hexyloxy, including their isomers. "Lower alkoxy" as used herein denotes an alkoxy group with a "lower alkyl" group as previously defined. "Ci-io alkoxy" as used herein refers to an-O-alkyl wherein alkyl is Ci-io.

The term “halogen” as used herein means fluorine, chlorine, bromine or iodine. In one embodiment, halogen may be chlorine. A “patient” or “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or rhesus monkey, and the terms “patient” and “subject” are used interchangeably herein.

The term “carrier”, as used in this disclosure, encompasses carriers, excipients, and diluents and means a material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body.

The term “treating”, with regard to a subject, refers to improving at least one symptom of the subject's disorder. Treating can be curing, improving, or at least partially ameliorating the disorder.

The term “disorder” is used in this disclosure to mean, and is used interchangeably with, the terms disease, condition, or illness, unless otherwise indicated.

The term “administer”, “administering”, or “administration” as used in this disclosure refers to either directly administering a compound or pharmaceutically acceptable salt of the compound or a composition to a subject, or administering a prodrug derivative or analog of the compound or pharmaceutically acceptable salt of the compound or composition to the subject, which can form an equivalent amount of active compound within the subject’s body.

The term “optionally substituted,” as used in this disclosure, means a suitable substituent can replace a hydrogen bound to a carbon, nitrogen, or oxygen. When a substituent is oxo (i.e., = O) then 2 hydrogens on the atom are replaced by a single O. In one embodiment, an alkyl or lower alkyl group can substituted with, for example, -N3, -C=CH, phenyl or OH. It will be understood by those skilled in the art, with respect to any group containing one or more substituents, that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically non-feasible and/or inherently unstable. Furthermore, combinations of substituents and/or variables within any of the Formulae represented herein are permissible only if such combinations result in stable compounds or useful synthetic intermediates wherein stable implies a reasonable pharmacologically relevant half-life at physiological conditions.

Dosage and Administration:

The compounds of the present invention may be formulated in a wide variety of oral administration dosage forms and carriers. Oral administration can be in the form of tablets, coated tablets, dragees, hard and soft gelatin capsules, solutions, emulsions, syrups, or suspensions. Compounds of the present invention are efficacious when administered by other routes of administration including continuous (intravenous drip) topical parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include a penetration enhancement agent), buccal, nasal, inhalation and suppository administration, among other routes of administration. The preferred manner of administration is generally oral using a convenient daily dosing regimen which can be adjusted according to the degree of affliction and the patient's response to the active ingredient.

A compound or compounds of the present invention, as well as their pharmaceutically useable salts, together with one or more conventional excipients, carriers, or diluents, may be placed into the form of pharmaceutical compositions and unit dosages. The pharmaceutical compositions and unit dosage forms may be comprised of conventional ingredients in conventional proportions, with or without additional active compounds or principles, and the unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. The pharmaceutical compositions may be employed as solids, such as tablets or filled capsules, semisolids, powders, sustained release formulations, or liquids such as solutions, suspensions, emulsions, elixirs, or filled capsules for oral use; or in the form of suppositories for rectal or vaginal administration; or in the form of sterile injectable solutions for parenteral use. A typical preparation will contain from about 5% to about 95% active compound or compounds (w/w). The term "preparation" or "dosage form" is intended to include both solid and liquid formulations of the active compound and one skilled in the art will appreciate that an active ingredient can exist in different preparations depending on the target organ or tissue and on the desired dose and pharmacokinetic parameters.

The term “excipient” as used herein refers to a compound that is useful in preparing a pharmaceutical composition, generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use. The compounds of this invention can be administered alone but will generally be administered in admixture with one or more suitable pharmaceutical excipients, diluents or carriers selected with regard to the intended route of administration and standard pharmaceutical practice.

“Pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.

A "pharmaceutically acceptable salt" form of an active ingredient may also initially confer a desirable pharmacokinetic property on the active ingredient which were absent in the non-salt form, and may even positively affect the pharmacodynamics of the active ingredient with respect to its therapeutic activity in the body. The phrase “pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxy ethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-l -carboxylic acid, glucoheptonic acid, 3 -phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier may be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component generally is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Suitable carriers include but are not limited to magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. Solid form preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

Liquid formulations also are suitable for oral administration include liquid formulation including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions. These include solid form preparations which are intended to be converted to liquid form preparations shortly before use. Emulsions may be prepared in solutions, for example, in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing, and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

The compounds of the present invention may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.

The compounds of the present invention may be formulated for topical administration to the epidermis as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also containing one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. Formulations suitable for topical administration in the mouth include lozenges comprising active agents in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

The compounds of the present invention may be formulated for administration as suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify.

The compounds of the present invention may be formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

The compounds of the present invention may be formulated for nasal administration. The solutions or suspensions are applied directly to the nasal cavity by conventional means, for example, with a dropper, pipette or spray. The formulations may be provided in a single or multidose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomizing spray pump.

The compounds of the present invention may be formulated for aerosol administration, particularly to the respiratory tract and including intranasal administration. The compound will generally have a small particle size for example of the order of five (5) microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC), for example, dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, or carbon dioxide or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by a metered valve. Alternatively the active ingredients may be provided in a form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). The powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of e.g., gelatin or blister packs from which the powder may be administered by means of an inhaler. When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient. For example, the compounds of the present invention can be formulated in transdermal or subcutaneous drug delivery devices. These delivery systems are advantageous when sustained release of the compound is necessary and when patient compliance with a treatment regimen is crucial. Compounds in transdermal delivery systems are frequently attached to a skin-adhesive solid support. The compound of interest can also be combined with a penetration enhancer, e.g., Azone (1-dodecylaza- cycloheptan-2-one). Sustained release delivery systems are inserted subcutaneously into to the subdermal layer by surgery or injection. The subdermal implants encapsulate the compound in a lipid soluble membrane, e.g., silicone rubber, or a biodegradable polymer, e.g., polylactic acid.

Suitable formulations along with pharmaceutical carriers, diluents and excipients are described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pennsylvania. A skilled formulation scientist may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering the compositions of the present invention unstable or compromising their therapeutic activity.

The modification of the present compounds to render them more soluble in water or other vehicle, for example, may be easily accomplished by minor modifications (salt formulation, esterification, etc.), which are well within the ordinary skill in the art. It is also well within the ordinary skill of the art to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in patients.

The term "therapeutically effective amount" as used herein means an amount required to reduce symptoms of the disease in an individual. The dose will be adjusted to the individual requirements in each particular case. That dosage can vary within wide limits depending upon numerous factors such as the severity of the disease to be treated, the age and general health condition of the patient, other medicaments with which the patient is being treated, the route and form of administration and the preferences and experience of the medical practitioner involved. For oral administration, a daily dosage of between about 0.01 and about 1000 mg/kg body weight per day should be appropriate in monotherapy and/or in combination therapy. A preferred daily dosage is between about 0.1 and about 500 mg/kg body weight, more preferred 0.1 and about 100 mg/kg body weight, and most preferred 1.0 and about 15 mg/kg body weight per day. Thus, for administration to a 70 kg person, the dosage range in one embodiment would be about 70 mg to .7 g per day. The daily dosage can be administered as a single dosage or in divided dosages, typically between 1 and 5 dosages per day. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect for the individual patient is reached. One of ordinary skill in treating diseases described herein will be able, without undue experimentation and in reliance on personal knowledge, experience and the disclosures of this application, to ascertain a therapeutically effective amount of the compounds of the present invention for a given disease and patient.

The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

Compounds of the present invention can be prepared beginning with commercially available starting materials and utilizing general synthetic techniques and procedures known to those skilled in the art. Chemicals may be purchased from companies such as for example SigmaAldrich, Argonaut Technologies, VWR and Lancaster. Chromatography supplies and equipment may be purchased from such companies as for example AnaLogix, Inc, Burlington, Wis.; Biotage AB, Charlottesville, Va.; Analytical Sales and Services, Inc., Pompton Plains, N.J.; Teledyne Isco, Lincoln, Nebr.; VWR International, Bridgeport, N.J.; and Waters Corporation, Milford, MA. Biotage, ISCO and Analogix columns are pre-packed silica gel columns used in standard chromatography.

EXAMPLES

The following examples further describe and demonstrate particular embodiments within the scope of the present invention. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). The disclosure is further illustrated by the following examples, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and/or scope of the appended claims.

Examplel

Synthesis Protocol of Compounds SC01 and SC18

Synthetic routes leading to compounds SC01 and SC 18 were developed and used to produce 100 mg of each compound in >95% purity. To complete the synthesis of compound SC01 (Figure 6, Scheme 1), the commercially available (2S, 4R)-4-(benzyloxy)-l-(tert- butoxycarbonyl)pyrro- lidine-2-carboxylic acid was esterified (ethyl iodide, DMF) (46) to form the corresponding ethyl ester, followed by acid-catalyzed removal of the N-Boc protecting group (HC1, dioxane) (47) to form the free amine intermediate. Addition of 3,4-dichlorobenzoic acid was accomplished using standard peptide coupling conditions (EDC, DIEA (48) followed by hydrogenolysis of the benzyl protecting group to afford the desired target compound ethyl (2S,4R)-l-(3,4- dichlorobenzoyl)-4- hydroxyl pyrrolidine-2-carboxylate, SC01. A series of homologues of SC01 can be readily synthesized by introducing chemical diversity into the synthesis through the use of substituted starting proline analogues or by introduction of variously substituted carboxylic acids in the final step (General Structure 1, X = O, N or S; Y = N, O or S; Rl, R2 = alkyl, aralkyl; R3, R4 = electron withdrawing group). The synthesis of SC 18 (Fig. A, Scheme 2), was accomplished in a single step by condensing methyl 2- amino-5-nitrobenzoate with (2-chloro-5- trifluoromethyl)- benzylisocyanate under microwave conditions (49). Homologues of SC 18 can be synthesized by introducing chemical diversity through the use of substituted starting anthranilic acid analogues or by introduction of variously substituted isocyanates or isothiocyanates (General Structure 2, X = O or S; Y, Z = electron withdrawing group; Rl - R5 = alkyl, aralkyl or electron withdrawing group).

Example!

Biological Examples and Results

Materials. Antimycin A, carbonyl cyanide 3 -chlorophenylhydrazone (CCCP), oligomycin, rotenone, zosuquidar, and tetramethylrhodamine methyl ester (TMRM), were purchased from Millipore Sigma (Burlington, MA). Fetal bovine serum (FBS) was from Atlanta Biologicals. Penicillin, streptomycin, 100X MEM nonessential amino acids, and RPMI 1640 containing 2.05 Mm L-glutamine, were from Thermo Fisher Scientific (Waltham, MA, USA). Eagle’s Minimum Essential Medium was from the American Type Culture Collection (ATCC; Manassas, VA, USA). All other chemicals were analytical grade.

Virtual screening. The inventors used a virtual screening strategy to identify novel compounds targeting the NADH-binding pocket. The inventors chose an NMR structure of VD AC (PDB : 6TIR), solved with NADH bound within the pore among the 12 X-ray or NMR structures resolved for VDAC (36). NADH binding reduces the conductance of the pore sterically without triggering a structural change. The inventors used the NADH pocket for the virtual screening of the South Carolina Compound Collection SC³ comprised of over 130,000 compounds, including 100,000 proprietary and fully annotated drug-like molecules. To optimize the screen, the inventors used the nicotinamide moiety and adjacent phosphate groups that is resolved in a well-defined VDAC pocket to generate shape-based queries, while omitting the adenosine moiety that freely rotates within the channel. The shape-based virtual screening, using the OpenEye software ROCS (OpenEye Scientific Software, Inc), assumes that compounds with similar shape and electrostatic characteristics will bind to the same target. The inventors next performed molecular docking of the top 20,000 compounds using the extra precision (XP) scoring function against VDAC1 (Glide; Schrodinger Inc). The top 250 scoring compounds were selected based on the lowest scoring ligand poses that predict the binding affinity and conformation of the ligand. Finally, 72 compounds were selected for physical screening using A m as a readout of mitochondrial metabolism.

Expression and purification of recombinant human VDAC1. pET-29a recombinant human VDAC1 (rhuVDACl) Open Reading Frame (ORF) clone plasmid construct, was purchased from GenScript and confirmed using sequencing. The plasmid was transformed into expression strain BL21 (DE3) (ThermoFisher) bacteria grown in Luria-Bertani media containing 50 pg/mL Kanamycin. The expression of the His-tagged rhuVDACl was induced by ImM isopropyl-P-d- thiogalactopyranoside (IPTG) for 4 h at 37° C with vigorous shaking. Bacteria were then harvested by centrifugation at 6000 rpm at 4° C for 10 min. The biomass (6-7 g/L) obtained was resuspended in buffer A (1 :50 v/v, 20mM Tris, pH 7.9, 200mM NaCl, 1 mM phenylmethyl sulfonyl fluoride (PMSF), EDTA free IX protease inhibitor cocktail). Resuspended bacteria were incubated with 0.5 mg/mL of lysozyme on ice for 30 min and lysed by pulse sonication. Inclusion bodies were washed in buffer A before solubilizing in buffer B (1 :20 v/v, 4 M guanidine-HCl, 20 mM Tris- HC1, pH 7.9, 500mM NaCl, and 10% glycerol) at 4° C for 30 min with gentle stirring. The supernatant containing the denatured rhuVDACl was collected by centrifugation at 15,000 g for 30 min. Buffer B (containing- 20 mg total soluble proteins) was incubated with 1 mL of Nickel- Nitriloacetic acid (Ni-NTA) Superflow Agarose beads; before incubation with 5 mL of denatured protein supernatant for 2 h, followed by washing with buffer B. Further 3 -step wash with buffer B mixed with buffer C (2% n-octyl-b-D-glucopyranoside (OG) from CalBiochem, 20 mM Tris, pH 7.9, 500 mM NaCl, and 10% glycerol) plus 10 mM imidazole (B:C = 1 :3, 1 :7, and 1 : 15, respectively) was performed to ensure maximal refolding and renaturing of the protein, and to remove excessive impurity. Additional washes with buffer C and 10 mM imidazole removed excess of guanidine, before the His-tagged rhuVDACl was eluted using buffer C supplemented with 300 mM imidazole under gravity at 4 °C. Protein fractions were dialyzed against buffer C for 4-6 h. Final purity and identity of the desired proteins were confirmed by analyzing the bands in SDS-PAGE and western blot.

Cell Culture. HepG2 (Cat # HB-8065) and SNU-449 (Cat # CRL-2234) human hepatocellular carcinoma, were purchased from the American Tissue Culture Collection (ATCC) (Manassas, VA). HepG2 cells were grown in Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) premium (Atlanta Biologicals), 100 units/mL penicillin and 100 pg/mL streptomycin. SNU-449 cells were grown in RPMI-1640 with the addition of 10% fetal bovine serum (FBS) premium (Atlanta Biologicals), 100 units/mL penicillin and 100 pg/ mL streptomycin. Both cell lines were maintained in 5% CCb/air at 37° C. All experiments were performed with cells at 70-80% confluency.

VDAC KO cells. sgRNA sequences targeting Exon 4 of VDAC 1, Exon 5 of VDAC2 and Exon 5 of VDAC3 were designed using Synthego (Synthego, Redwood City, CA) or CRISPOR (http://crispor.tefor.net/) and synthesized by IDT (IDT, San Diego, CA). sgRNA sense and antisense strands were: VDAC1, ACTAGGCACCGAGATTACTG and

CAGTAATCTCGGTGCCTAGT; VDAC2, CGCGCGTCGTAAGTAAAGCT and AGCTTTACTTACGACGCGCG; VDAC3, GACCAGAAGTAGAAAATTCC and GGAATTTTCT ACTTCTGGTC .

Each of the guide RNA sequences were cloned into the PX459 pSPCas9(BB)-2A-Puro vector. For transfection, HepG2 cells were plated at approximately 50% confluence in 10 cm tissue culture dishes. The following day, cells were transfected with 30 pg of total sgRNA, 15 pg each, in the following combinations: VDAC1/VDAC2, VDAC1/VDAC3, and VDAC2/ VDAC3 using Viafect (Promega, Cat. # E4981), according to the manufacturer’s protocol. After 24 h, the media was changed to EMEM 10% FBS + puromycin 1 ug/ul for 48-72 hours, until all cells in a negativecontrol plate treated with selection media were dead. Following selection, cells were maintained in EMEM 10% FBS and allowed to grow until colonies were visible. Colonies were then transferred into each well of a 96-well plate and allowed to grow for an additional 2-3 d. Colonies were dissociated with trypsin and divided between two 96 well plates. After 2-3 d of growth, the wells of one plate were harvested using 50 pl QuickExtract (QuickExtract, Middleton, WI), following the manufacturer’s instructions. Extracted DNA was used for PCR with primers designed to amplify the target region. Forward and reverse primers were: VDAC1, GTGCAGGCTGTGACTCTTCT and AAGGTCAGCTTCAGTCCACG; VDAC2, AGGGAGGAAGGAAGCTGTCTGC and GTCACAAAGGGCTTCCACCACC; VDAC3, GCTGGTCTTGAGCTCCTGGACT and ACCCAGGAGAAGCTTAGCTGTGT.

Following PCR amplification, the products were digested with enzymes with recognition sites near the sgRNA cut site (VDAC1 - Mbol, VDAC2 - Alul, and VDAC3 - Apol). If CRISPR-Cas9 modification was successful, the enzyme would fail to digest the PCR product. When potential hits were identified, PCR products of the target regions were sequenced and ICE analysis (Synthego), was used to identify and confirm knockout success.

Confocal microscopy of tetramethyl rhodamine methylester (TMRM) and NADH. HepG2 and SNU-449 cells were plated in Grenier Bio-One TC 4-chamber plates (Greiner-Bio-One, Monroe, NC), or 35 mm MatTek dishes (MatTek Corporation, Ashland, MA). Cells were loaded with 20 nM TMRM for 60 min in modified Hank's balanced salt solution (HBSS) containing (in mM): NaCl 137, Na₂HPO₄ 0.35, KC1 5.4, KH₂PO₄ 1, MgSO₄ 0.81, Ca₂Cl 0.95, glucose 5.5, NaHCOs 25 and HEPES 20, pH 7.4. Live cells, maintained in the Zeiss LSM 880 NLO inverted laser scanning confocal microscope (Thornwood, NY) environmental chamber (humidified 5% CO₂ at 37° C), were imaged with a 63X 1.4 N. A. plan apochromat oil immersion lens. TMRM was excited at 561 nm. Emission was detected with a Quasar multichannel spectral detector at 590- 610 nm through a one Airy unit diameter pinhole. TMRM intensities were quantified to make relative comparisons using Photoshop CS4 software (Adobe Systems, San Jose, CA) as previously described (27, 37). A minimum of 4 randomly selected fields with 8-20 cells per field were imaged during the time course of 3 independent experiments for all microscopy experiments.

NADH autofluorescence was imaged using multiphoton laser excitation (720 nm, 3% power) and an infrared-blocking emission barrier filter (460 ±25 nm). TMRM and NADH intensity were quantified using Zeiss Zen and Photoshop CS4 (Adobe Systems, San Jose, CA, USA) software after subtraction of background fluorescence as previously described (38).

ATP measurement. ATP was determined in cells lysed with the luciferase-based Cell Titer-Gio Luminescent Assay Kit (Promega, WI, Cat # G7570). Luminescence was measured in a Biotek Synergy Hl plate reader. All luminescence values were normalized per million cells, and expressed as percentage compared to vehicle.

Respirometric assay. Oxygen consumption rates (OCR) were measured using a Seahorse XF96 analyzer (Agilent Technologies, Santa Clara, CA, USA), and calculated from the continuous average slope of the O2 partitioning among plastic, atmosphere, and cellular uptake (39, 40). SNU- 449 or HepG2 cells (25,000/ well) in XF 96 well microplates were maintained in growth media and treated 24 h after plating. Experiments were performed in 200 pL/ well of respiratory substrate (RS) buffer containing (in mM): L-glutamine 4, D-Glucose 10, sodium pyruvate 1, CaCL 0.036, MgCh 0.06, KH2PO4 0.05, KC1 0.54, Na₂HPO₄ heptahydrate 0.05, HEPES 2 and NaCl 13 (pH: 7.4). Microplates and sensor cartridges were kept in an air incubator for 1 h before starting the experiments. Basal, oligomycin sensitive, and maximal respiratory capacity were determined after sequential addition of OLIGO (IpM), CCCP (1 pM), and rotenone (2 pM) + antimycin A (2 pM), respectively.

Wound healing. SNU-449 cells were plated in 2-wells Ibidi Culture-inserts (Grafelfing, Germany). After overnight incubation, inserts were removed and cells were washed with PBS. Fresh medium containing SC18, sorafenib, or vehicle was then added to the culture plate, which was incubated using a humidified 5% CO2/ air at 37° C in a BIOTEK Cytation 5 multimode (imaging) reader equipped with a BioSpa automated incubator and MultiFlo liquid handler (Agilent, CA, USA). The entire plate was imaged in a time-lapse mode every 4 h for 2 d.

EC50. SNU-449 cells plated in glass bottom, 10-well Cellview culture slides (Greiner-Bio-One), were incubated with 20 nM TMRM in HBSS for 1 h. EC50 was calculated assessing mean intensity of TMRM fluorescence (GraphPad Prism, San Diego, CA) after 2 h exposure to SC18 at increasing concentrations.

LC50. SNU-449 cells plated in 6 well tissue culture plates (VWR, Radnor, NC) were exposed to SC18 at increasing concentrations for 5 d. Cell viability was determined using a trypan blue exclusion assay and calculated using GraphPad Prism.

Statistics. Statistical differences between groups were analyzed by the Student’s t-test using p < 0.05 as the criterion of significance. Data points are the means ± S.E. of 3-5 independent experiments. Images in figures are representative of three or more independent experiments unless otherwise stated.

RESULTS

Identification of SC 18

The inventors screened in silico the South Carolina Compound Collection SC³, using a shapebased query, as described in Material & Methods. The inventors chose the nicotinamide moiety, that interacts directly with the NADH binding pocket, and the sugar ring attached to a phosphate molecule, omitting the adenine group that sterically occludes the VDAC1 pore (Fig 1.). The in silico screen yielded 72 compounds with the lowest scoring ligand poses that predict the binding affinity and conformation of the ligand (Fig 1.). The 72 compounds were further tested for their ability to induce changes in TMRM fluorescence, and indicator of A m, used as a readout of overall mitochondrial metabolism. The inventors established an arbitrary threshold of 40% change in TMRM fluorescence compared to baseline, as a criterion to select hits. The inventors identified SC18 as a most potent hit using this screening strategy (Fig. 2A). SC18 binds to VDAC

To determine binding to the target, the inventors used both an in silico and a wet-lab approach. The inventors overlaid in silico SC18 with P-NADH, in the NADH binding pocket of VDAC 1 (NMR structure, 6TIR). The predicted binding pose of SC18 indicates that it may bind to the NADH-binding pocket in a very similar orientation compared to P-NADH.

To determine if SC18 bound to VDAC, the inventors incubated SC18 with recombinant VDAC 1 purified from E. Coli (41), and performed an isothermal calorimetric assay with P-NADH as positive control, using a NanoTemper Tycho NT. 6 instrument. SC18 addition to VDAC1, similar to P-NADH, caused a temperature shift of ~7 °C indicating that SC18 binds to VDAC1 (Fig. 2B).

For clarity on the binding of SC18, a transparent surface is shown for NADH bound to the NMR structure of VDAC (Fig. 2C). The quinazolinedione scaffold forms two hydrogen bonds with lysine 20, one with the backbone and the terminal nitrogen; and other a similar interaction to the ring oxygen from the sugar ring closest to the 1, 4 dihydronicotinamide moiety of NADH. The two pi-cation interactions were found between the quinazoline-dione and Arginine 218. The 6-nitro substitution formed a hydrogen bond with Tyrosine 22, similar to the phosphate backbone of NADH. Finally, the trifluoromethyl overlays with an amide moiety of the 1,4-dihydronicotinamide of NADH in the binding pocket.

SC18 modulates mitochondrial metabolism

Mitochondrial membrane potential. To initially assess the effects of SC18 on mitochondrial metabolism, the inventors quantitatively assessed A m and determined the EC50. A m is an overall indicator of mitochondrial metabolism. The inventors loaded HepG2 and SNU449 cells with the fluorophore potential indicator TMRM, and imaged TMRM fluorescence by confocal microscopy. Baseline A m was similar in wild type and cells treated with vehicle (Fig. 3A, and not shown). Treatment with SC18 for 2 h, decreased AFm by ~ 50% compared to vehicle. To determine if the drop in A m caused by SC18 was differentially affected by a specific VDAC isoform, the inventors used SC18 at the same concentration in wild type HepG2, and in VDAC 1/2, VDAC 1/3 and VDAC2/3 double KO cells. SC18 (25 pM) decreased similarly AFm in Wt, VDAC 1/2, 2/3, and 1/3 double KO cells (~ 52%, 46%, 61%, and 44 % respectively) indicating that SC18 was targeting all VDAC isoforms (Fig. 3A and B.). EC50 (25pM) (Fig. 3C), was calculated based on changes in A m, as described in Material & Methods.

NADH generation. Mitochondrial NADH is an indicator of the balance between oxidation of respiratory substrates in the Krebs cycle that reduce NAD+ to NADH, and the utilization by the respiratory chain. To assess whether SC 18 influenced mitochondrial NADH generation, the inventors assessed NADH autofluorescence by multiphoton fluorescence microscopy. SC 18 for 2 h decreased mitochondrial NADH by - 19% in HepG2 cells (Fig. 4A-B).

Respiration. Since changes in A m may or may not be associated with differences in oxidative phosphorylation, the inventors determined cellular respiration (Oxygen consumption rates) in SNU449 cells, using a Seahorse XFe96 extracellular flux analyzer. Cells were incubated in the presence of 1, 3, 10, or 30 pM SC 18 for 2 h . SC 18 increased basal respiration in a dose-dependent fashion (-154 pmol/ min/ 3.5 X 10⁴ cells) to a maximum of~243 pmol/ min/ 3.5 X 10⁴ cells at 25 pM) (Fig. 4C).

ATP production. To determine if SC18 also influenced ATP production, the inventors measured cellular ATP using a luminescence assay. SC18 (25 pM) for 2 h, decreased ATP by - 24%; indicating that the increase in respiration was not coupled to a higher synthesis of ATP (Fig. 4D).

SC 18 is cytotoxic To determine if long-term exposure to SC18 would inhibit cell proliferation and eventually cause cell death, the inventors assessed cell viability using a trypan blue exclusion assay. SC18 decreased SNU-449 cell viability by more than 50% at concentrations higher than 24 pM for 5 d. Based on this method to assess viability, the inventors determined an LC50 of 24 pM for SC18 (Fig. 5 A).

SC18 delays wound healing.

To confirm if cell viability was associated also with a decrease in cell migration and proliferation, the inventors used a scratch assay. Images were taken at 8 h intervals up to 48 h after treatment with SC18 (24 pM) (Fig. 5B and C). Sorafenib, a chemotherapeutic agent that promote hepatocarcinoma cell death, was used as a positive control. SC18 delayed wound healing similar to SOR (5 pM). The maximal inhibitory effect (~50% inhibition), occurred in the interval between 16 and 32 h after treatment. 48 h after treatment, the inhibition effect of SC18 on wound healing was still ~30%.

In summary, the findings show that SC 18 as a representative compound of the invention modulates mitochondrial metabolism in cancer cells, inhibits cell proliferation and induces cell death.

The invention is further described in the following numbered paragraphs:

1. A compound of formula (I):

wherein: X is O or S;

Y and Z are, individually, an electron withdrawing group; and

R1 to R5 are, individually, alkyl, aralkyl or an electron withdrawing group; or a pharmaceutically acceptable salt thereof.

2. A compound of formula (la):

(la), wherein:

Ri is hydrogen or -C(O)O-lower alkyl;

R2 is hydrogen, -CF3 or -NO2;

R3 is hydrogen, halogen or lower alkyl;

R4 is hydrogen or lower alkyl;

R5 is hydrogen or halogen;

Rs is hydrogen; and R? is hydrogen, halogen, -NO2, -NH2, or a pharmaceutically acceptable salt thereof.

3. The compound according to paragraph 2, wherein said compound is:

or a pharmaceutically acceptable salt individually thereof.

4. A pharmaceutical composition, comprising a therapeutically effective amount of a compound according to paragraph 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

5. A method for inducing mitochondrial dysfunction, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a compound according to paragraph 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. A method for inhibiting cell proliferation, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a compound according to paragraph 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

7. A method for the treatment of cancer, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a compound according to paragraph 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. REFERENCES

1. Warburg O. Ueber den stoffwechsel der tumoren. London: Constable; 1930 1930.

2. Warburg O, Wind F, Negelein E. THE METABOLISM OF TUMORS IN THE BODY. J Gen Physiol. 1927;8(6):519-30.

3. Lim HY, Ho QS, Low J, Choolani M, Wong KP. Respiratory competent mitochondria in human ovarian and peritoneal cancer. Mitochondrion. 2011;11(3):437-43. doi: S1567- 7249(10)00235-7 [pii]; 10.1016/j .mito.2010.12.015 [doi],

4. Maldonado EN, Patnaik J, Mullins MR, Lemasters JJ. Free tubulin modulates mitochondrial membrane potential in cancer cells. Cancer Res. 2010;70(24): 10192-201. Epub 2010/12/17. doi: 10.1158/0008-5472.CAN-10-2429. PubMed PMID: 21159641; PMCID: PMC3010233.

5. Mathupala SP, Ko YH, Pedersen PL. The pivotal roles of mitochondria in cancer: Warburg and beyond and encouraging prospects for effective therapies. Biochim Biophys Acta. 2010;1797(6-7):1225-30. doi: S0005-2728(10)00131-3 [pii]; 10.1016/j .bbabio.2010.03.025 [doi],

6. Moreno- Sanchez R, Marin-Hernandez A, Saavedra E, Pardo JP, Ralph SJ, Rodriguez- Enriquez S. Who controls the ATP supply in cancer cells? Biochemistry lessons to understand cancer energy metabolism. Int J Biochem Cell Biol. 2014;50:10-23. doi: S1357-2725(14)00042-9 [pii]; 10.1016/j .biocel.2014.01.025 [doi],

7. Nakashima RA, Paggi MG, Pedersen PL. Contributions of glycolysis and oxidative phosphorylation to adenosine 5'-triphosphate production in AS-30D hepatoma cells. Cancer Res. 1984;44(12 Pt l):5702-6.

8. Singleterry J, Sreedhar A, Zhao Y. Components of cancer metabolism and therapeutic interventions. Mitochondrion. 2014;17C:50-5. doi: S1567-7249(14)00084-l

[pii]; 10.1016/j. mito.2014.05.010 [doi],

9. DeBerardinis RJ, Sayed N, Ditsworth D, Thompson CB. Brick by brick: metabolism and tumor cell growth. Curr Opin Genet Dev. 2008; 18(1):54-61. doi: S0959-437X(08)00028-2 [pii]; 10.1016/j .gde.2008.02.003 [doi] .

10. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324(5930):1029-33. doi: 324/5930/1029 [pii]; 10.1126/science.1160809 [doi],

11. DeBerardinis RJ, Chandel NS. We need to talk about the Warburg effect. Nat Metab. 2020'2(2): 127-9. Epub 2020/07/23. doi: 10.1038/s42255-020-0172-2. PubMed PMID: 32694689.

12. Liemburg-Apers DC, Schirris TJ, Russel FG, Willems PH, Koopman WJ. Mitoenergetic Dysfunction Triggers a Rapid Compensatory Increase in Steady-State Glucose Flux. Biophys J. 2015;109(7): 1372-86. doi: S0006-3495(15)00784-5 [pii]; 10.1016/j bpj .2015.08.002 [doi],

13. Robinson GL, Dinsdale D, Macfarlane M, Cain K. Switching from aerobic glycolysis to oxidative phosphorylation modulates the sensitivity of mantle cell lymphoma cells to TRAIL. Oncogene. 2012;31(48):4996-5006. Epub 2012/02/09. doi: 10.1038/onc.2012.13. PubMed PMID: 22310286. 14. Smolkova K, Bellance N, Scandurra F, Genot E, Gnaiger E, Plecita-Hlavata L, Jezek P, Rossignol R. Mitochondrial bioenergetic adaptations of breast cancer cells to aglycemia and hypoxia. J Bioenerg Biomembr. 2010;42(l):55-67. doi: 10.1007/sl0863-009-9267-x [doi],

15. De P, V, Guarino F, Guarnera A, Messina A, Reina S, Tomasello FM, Palermo V, Mazzoni C. Characterization of human VDAC isoforms: a peculiar function for VDAC3? Biochim Biophys Acta. 2010;1797(6-7): 1268-75. doi: 80005-2728(10)00042-3 [pii]; 10.1016/j .bbabio.2010.01.031 [doi],

16. Sampson MJ, Lovell RS, Craigen WJ. The murine voltage-dependent anion channel gene family. Conserved structure and function. J Biol Chem. 1997;272(30): 18966-73.

17. Hiller S, Abramson J, Mannella C, Wagner G, Zeth K. The 3D structures of VDAC represent a native conformation. Trends Biochem Sci. 2010;35(9):514-21. doi: S0968- 0004(10)00049-6 [pii]; 10.1016/j .tibs.2010.03.005 [doi],

18. Ujwal R, Cascio D, Colletier JP, Faham S, Zhang J, Toro L, Ping P, Abramson J. The crystal structure of mouse VDAC1 at 2.3 A resolution reveals mechanistic insights into metabolite gating. Proc Natl Acad Sci U S A. 2008; 105(46): 17742-7. doi: 0809634105 [pii]; 10.1073/pnas.0809634105 [doi],

19. Bowen KA, Tam K, Colombini M. Evidence for titratable gating charges controlling the voltage dependence of the outer mitochondrial membrane channel, VDAC. J Membr Biol. 1985;86(1):51-9. Epub 1985/01/01. doi: 10.1007/BF01871610. PubMed PMID: 2413210.

20. Colombini M. Voltage gating in the mitochondrial channel, VDAC. J Membr Biol. 1989;11 l(2): 103-l 1.

21. Choudhary OP, Ujwal R, Kowallis W, Coalson R, Abramson J, Grabe M. The electrostatics of VDAC: implications for selectivity and gating. J Mol Biol. 2010;396(3):580-92. doi: S0022- 2836(09)01476-4 [pii]; 10.1016/j .jmb.2009.12.006 [doi],

22. Colombini M. The VDAC channel: Molecular basis for selectivity. Biochim Biophys Acta. 2016;1863(10):2498-502. Epub 2016/01/31. doi: 10.1016/j.bbamcr.2016.01.019. PubMed PMID: 26826035.

23. Villinger S, Giller K, Bayrhuber M, Lange A, Griesinger C, Becker S, Zweckstetter M. Nucleotide interactions of the human voltage-dependent anion channel. J Biol Chem. 2014;289(19): 13397-406. Epub 2014/03/29. doi: 10.1074/jbc.Ml 13.524173. PubMed PMID: 24668813; PMCID: PMC4036348.

24. Colombini M. VDAC structure, selectivity, and dynamics. Biochim Biophys Acta. 2012; 1818(6): 1457-65. Epub 2012/01/14. doi: 10.1016/j .bbamem.2011.12.026. PubMed PMID: 22240010; PMCID: PMC3327780.

25. Tan W, Colombini M. VDAC closure increases calcium ion flux. Biochim Biophys Acta. 2007; 1768(10):2510-5. doi: 80005-2736(07)00213-1 [pii]; 10.1016/j .bbamem.2007.06.002 [doi],

26. Sander P, Gudermann T, Schredelseker J. A Calcium Guard in the Outer Membrane: Is VDAC a Regulated Gatekeeper of Mitochondrial Calcium Uptake? Int J Mol Sci. 2021;22(2). Epub 2021/01/23. doi: 10.3390/ijms22020946. PubMed PMID: 33477936; PMCID: PMC7833399.

27. Maldonado EN, Sheldon KL, DeHart DN, Patnaik J, Manevich Y, Townsend DM, Bezrukov SM, Rostovtseva TK, Lemasters JJ. Voltage-dependent anion channels modulate mitochondrial metabolism in cancer cells: regulation by free tubulin and erastin. J Biol Chem. 2013 ;288(17): 11920-9. doi: M112.433847 [pii]; 10.1074/jbc.Ml 12.433847 [doi],

28. Rostovtseva TK, Sheldon KL, Hassanzadeh E, Monge C, Saks V, Bezrukov SM, Sackett DL. Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration. Proc Natl Acad Sci U S A. 2008;105(48):18746-51. doi: 0806303105 [pii]; 10.1073/pnas.0806303105 [doi],

29. Timohhina N, Guzun R, Tepp K, Monge C, Varikmaa M, Vija H, Sikk P, Kaambre T, Sackett D, Saks V. Direct measurement of energy fluxes from mitochondria into cytoplasm in permeabilized cardiac cells in situ: some evidence for Mitochondrial Interactosome. J Bioenerg Biomembr. 2009;41(3):259-75. doi: 10.1007/sl0863-009-9224-8 [doi],

30. Al Jamal JA. Involvement of porin N,N-dicyclohexylcarbodiimide-reactive domain in hexokinase binding to the outer mitochondrial membrane. Protein J. 2005;24(l): l-8. Epub 2005/03/11. doi: 10.1007/sl0930-004-0600-2. PubMed PMID: 15756812.

31. Pastorino JG, Shulga N, Hoek JB. Mitochondrial binding of hexokinase II inhibits Bax- induced cytochrome c release and apoptosis. J Biol Chem. 2002;277(9):7610-8.

32. Tsujimoto Y, Shimizu S. VDAC regulation by the Bcl-2 family of proteins. Cell Death Differ. 2000;7(12): 1174-81. Epub 2001/02/15. doi: 10.1038/sj.cdd.4400780. PubMed PMID: 11175254.

33. Baines CP, Kaiser RA, Sheiko T, Craigen WJ, Molkentin JD. Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death. Nat Cell Biol. 2007;9(5):550-5.

34. Das S, Wong R, Rajapakse N, Murphy E, Steenbergen C. Glycogen synthase kinase 3 inhibition slows mitochondrial adenine nucleotide transport and regulates voltage-dependent anion channel phosphorylation. Circ Res. 2008;103(9):983-91. doi: CIRCRESAHA.108.178970 [pii] ; 10.1161/CIRCRES AHA.108.178970 [doi] .

35. Vander Heiden MG, Li XX, Gotti eib E, Hill RB, Thompson CB, Colombini M. Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane. J Biol Chem. 2001;276(22): 19414-9.

36. Bohm R, Amodeo GF, Murlidaran S, Chavali S, Wagner G, Winterhalter M, Brannigan G, Hiller S. The Structural Basis for Low Conductance in the Membrane Protein VDAC upon beta- NADH Binding and Voltage Gating. Structure. 2020;28(2):206-14 e4. Epub 2019/12/22. doi: 10.1016/j.str.2019.11.015. PubMed PMID: 31862297.

37. Christie CF, Fang D, Hunt EG, Morris ME, Rovini A, Heslop KA, Beeson GC, Beeson CC, Maldonado EN. Statin-dependent modulation of mitochondrial metabolism in cancer cells is independent of cholesterol content. FASEB J. 2019:fj201802723R. Epub 2019/04/06. doi: 10.1096/fj.201802723R. PubMed PMID: 30951369.

38. Maldonado EN, DeHart DN, Patnaik J, Klatt SC, Beck GM, Lemasters JJ. ATP/ADP Turnover and Import of Glycolytic ATP into Mitochondria in Cancer Cells is Independent of the Adenine Nucleotide Translocator. J Biol Chem. 2016. doi: Ml 16.734814 [pii]; 10.1074/jbc.Ml 16.734814 [doi],

39. Beeson CC, Beeson GC, Schnellmann RG. A high-throughput respirometric assay for mitochondrial biogenesis and toxicity. Anal Biochem. 2010;404(l):75-81. doi: 10.1016/j.ab.2010.04.040. PubMed PMID: 20465991; PMCID: PMC2900494. 40. Gerencser AA, Neilson A, Choi SW, Edman U, Yadava N, Oh RJ, Ferrick DA, Nicholls DG, Brand MD. Quantitative microplate-based respirometry with correction for oxygen diffusion. Anal Chem. 2009;81(16):6868-78. doi: 10.1021/ac900881z. PubMed PMID: 19555051; PMCID: PMC2727168.

41. Shi Y, Jiang C, Chen Q, Tang H. One-step on-column affinity refolding purification and functional analysis of recombinant human VDAC1. Biochem Biophys Res Commun. 2003;303(2):475-82. Epub 2003/03/28. doi: 10.1016/s0006-291x(03)00359-0. PubMed PMID: 12659842.

42. Fang D, Maldonado EN. VDAC Regulation: A Mitochondrial Target to Stop Cell Proliferation. Adv Cancer Res. 2018;138:41-69. Epub 2018/03/20. doi: 10.1016/bs.acr.2018.02.002. PubMed PMID: 29551129; PMCID: PMC6234976.

43. Heslop KA, Milesi V, Maldonado EN. VDAC Modulation of Cancer Metabolism: Advances and Therapeutic Challenges. Front Physiol. 2021;12:742839. Epub 2021/10/19. doi: 10.3389/fphys.2021.742839. PubMed PMID: 34658929; PMCID: PMC8511398.

44. Maldonado EN. VDAC-Tubulin, an Anti-Warburg Pro-Oxidant Switch. Front Oncol. 2017;7:4. Epub 2017/02/09. doi: 10.3389/fonc.2017.00004. PubMed PMID: 28168164; PMCID: PMC5256068.

45. Rosencrans WM, Rajendran M, Bezrukov SM, Rostovtseva TK. VDAC regulation of mitochondrial calcium flux: From channel biophysics to disease. Cell Calcium. 2021;94: 102356. Epub 2021/02/03. doi: 10.1016/j. ceca.2021.102356. PubMed PMID: 33529977; PMCID: PMC7914209.

46. DeHart DN, Fang D, Heslop K, Li L, Lemasters JJ, Maldonado EN. Opening of voltage dependent anion channels promotes reactive oxygen species generation, mitochondrial dysfunction and cell death in cancer cells. Biochem Pharmacol. 2018;148: 155-62. Epub 2018/01/01. doi: 10.1016/j .bcp.2017.12.022. PubMed PMID: 29289511; PMCID: PMC5909406.

47. DeHart DN, Lemasters JJ, Maldonado EN. Erastin-Like Anti-Warburg Agents Prevent Mitochondrial Depolarization Induced by Free Tubulin and Decrease Lactate Formation in Cancer Cells. SLAS Discov. 2018;23(l):23-33. Epub 2017/10/13. doi: 10.1177/2472555217731556. PubMed PMID: 29024608; PMCID: PMC5927820.

It is to be understood that the invention is not limited to the particular embodiments of the invention described above, as variations of the particular embodiments may be made and still fall within the scope of the appended claims.

Claims

CLAIMS:

1. A compound of formula (I):

wherein:

X is O or S;

Y and Z are, individually, an electron withdrawing group; and

R1 to R5 are, individually, alkyl, aralkyl or an electron withdrawing group; or a pharmaceutically acceptable salt thereof.

2. A compound of formula (la):

wherein:

Ri is hydrogen or -C(O)O-lower alkyl;

R2 is hydrogen, -CF3 or -NO2;

R3 is hydrogen, halogen or lower alkyl;

R4 is hydrogen or lower alkyl;

R5 is hydrogen or halogen;

Rs is hydrogen; and

R7 is hydrogen, halogen, -NO2, -NH2, or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 2, wherein said compound is:

or a pharmaceutically acceptable salt individually thereof.

4. A pharmaceutical composition, comprising a therapeutically effective amount of a compound according to claim 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

5. A method for inducing mitochondrial dysfunction, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a compound according to claim 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. A method for inhibiting cell proliferation, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a compound according to claim 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

7. A method for the treatment of cancer, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a compound according to claim 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.