CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority from Israel Patent Application No. 247025, filed on July 31, 2016. The content of the above document is incorporated by reference in its entirety as if fully set forth herein.

FIELD OF THE INVENTION

[002] The present invention, in some embodiments thereof, relates to small molecules (e.g., naphthoquinone derivatives) and using same for e.g., selective targeting of proteins that are overexpressed in cells (e.g., cancer cells).

BACKGROUND OF THE INVENTION

[003] Reactive oxygen species (ROS) homeostasis is important for the survival and progression of both normal and cancerous cells. Certain amounts of ROS are required for proper functioning, including normal metabolism and signaling, but excessive amounts lead to oxidative stress— the imbalance between the production of ROS and the elimination thereof by molecules or enzymes with antioxidant activity. Extreme oxidative stress will lead to complete cell death, as in the case of treatment of tumors by photodynamic therapy (PDT), but the effect of mild conditions is much less predictable. The outcome depends very much on the primary target that will be modified by reacting with the ROS including lipids, DNA, proteins, particular enzymes, and more.

[004] Overexpression of the ubiquitination-counteracting deubiquitinases (DUBs), a subclass of cysteine proteases, is documented in several disease states like cancer, neurodegenerative and viral disease.

SUMMARY OF THE INVENTION

[005] The present invention, in some embodiments thereof, relates to compounds comprising naphthoquinone derivatives and uses thereof for selective targeting of proteins that are overexpressed in cells (e.g., cancer cells). The disclosed compounds may induce the formation of reactive oxygen species (ROS), thereby assisting the therapeutic activity. [006] According to an aspect of some embodiments of the present invention, there is provided a compound represented by the structure of general formula (I) or a pharmaceutically acceptable salt thereof, for treating cancer.

wherein: (a) R ¹ is C1-C4 alkyl; (b) substituents R ² to R ⁶ , in each instance, comprise or are selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroalicyclic, heteroaryl, alkoxy, hydroxy, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amide, carboxy, sulfonyl, sulfoxy, sulfinyl, sulfonamide, and (c) wherein at least one substituent selected from R ³ to R ⁶ is alkoxy.

[007] In some embodiments, R ² is hydrogen. In some embodiments, R ¹ is methyl. In some embodiments, one or more substituents are selected from R ⁴ to R ⁵ is alkoxy. In some embodiments, each R ³ and R ⁶ is hydrogen.

[008] In some embodiments, either R ⁴ or R ⁵ is alkoxy. In some embodiments, the alkoxy is methoxy.

[009] In some embodiments, the compound is in the form of Formula IA:

[010] In some embodiments, the compound is in the form of Formula IB

[011] In some embodiments, the cancer is selected from the group consisting of: carcinoma, lymphoma, glioblastoma, and leukemia. In some embodiments, the carcinoma is selected from the group consisting of: breast cancer, and prostate cancer.

[012] According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising the compound of formula (I) and a pharmaceutically acceptable carrier or excipient.

[013] In some embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable agent selected from one or more of a stabilizer, a preservative, a chelating agent, a viscosity modifying agent, a buffering agent, and pH adjusting agent.

[014] In some embodiments, the pharmaceutical composition is formulated for parenteral, mucosal, nasal or oral administration.

[015] According to an aspect of some embodiments of the present invention, there is provided a use of the disclosed compound according to any embodiment thereof, or the disclosed pharmaceutical composition according to any embodiment thereof, for the manufacture of a medicament for treating a disease or disorder selected from the group consisting of: cancer, neurodegenerative disease or viral disease.

[016] In some embodiments, the disease or disorder is associated with overexpression of cysteine protease.

[017] In some embodiments, the cysteine protease is selected from ubiquitination- counteracting deubiquitinases (DUBs).

[018] According to an aspect of some embodiments of the present invention, there is provided a method comprising: identifying a subject having overexpression of cysteine protease- associated disease and administering to the subject an effective amount of the disclosed compound in any embodiment thereof.

[019] According to an aspect of some embodiments of the present invention, there is provided a process for the preparation of a compound of the Formula II

In some embodiments, the process comprises

contacting a compound of Formula III

(b) followed by reaction with R^OH and a metal chloride, to give Formula II, wherein:

(i) R ¹ is C1-C4 alkyl; (ii) substituents from R ³ to R ⁶ , in each instance, comprise or are selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroalicyclic, heteroaryl, alkoxy, hydroxy, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amide, carboxy, sulfonyl, sulfoxy, sulfinyl, or sulfonamide, and (iii) wherein at least one substituent selected from R ³ to R ⁶ is alkoxy.

[021] In some embodiments, R ¹ is methyl, and R ⁶ is hydrogen. In some embodiments, R ³ is hydrogen. In some embodiments, either R ⁴ or R ⁵ is alkoxy. In some embodiments, the alkoxy is methoxy.

[022] In some embodiments, the organic solvent is Dimethyl sulfoxide (DMSO). [023] In some embodiments, the hyper-valent iodine reagent is selected from phenyliodine diacetate (PIDA), [bis(trifluoroacetoxy)iodo]benzene (PIFA), Dess-Martin periodinane (DMP), and 2-iodoxybenzoic acid (IBX).

[024] In some embodiments, the hyper-valent iodine reagent and the compound of the Formula III are in molar ratio of from 3.5 to 4.5, respectively.

[025] In some embodiments, step (b) further comprises the presence of an oxidizing agent.

[026] In some embodiments, the oxidizing agent is sodium iodate.

[027] In some embodiments, step (b) further comprises presence of metal chloride.

[028] In some embodiments, the reaction of step (a) is conducted at a temperature range of about 10°C to about 50°C.

[029] In some embodiments, steps (a) and (b) are conducted in one pot.

[030] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[031] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[032] In the drawings:

[033] Figure 1 presents the disclosed compound, referred to as: compounds 1-24 hereinthroughout .

[034] Figures 2A-B present a scheme showing the synthesis of 1,2-naphthoquinones with different substituents on C4 (compound 1 to 7; Figure 2A), and the ^ NMR and ¹³ C-NMR spectra of compounds 1-3, and 5-7 (Figure 2B) (NMR: nuclear magnetic resonance). [035] Figures 3A-B present synthesis scheme of naphthoquinone derivatives (compounds 8- 14) starting from tetralone derivatives (Figure 3A) and the ^-NMR and ¹³ C-NMR spectra of compounds 8-13 (Figure 3B).

[036] Figure 4 presents comparison of USP2 inhibition capability by compounds with either ortho- or /?ara-quinone moieties, at either 1 or 5 μΜ concentrations.

[037] Figure 5A-B present a graph showing a plot of the inactivation rate constants (£ ₀ bs) vs. the concentration of compound 12 (nM), for obtaining the maximal rate of enzyme inactivation (kinact) (Figure 5A; each value represents the mean +SE of two independent experiments); and Mass spectrometry spectra of USP2 treated with A) upper panel: DMSO and B) lower panel: compound 12 for 15 min (Figure 5B). DMSO: dimethyl sulfoxide.

[038] Figure 6 presents schematic representation of redox cycling by o/ /zo-quinones and their mode of inhibition of DUBs via oxidation of the catalytic cys mainly to sulfinic acid.

[039] Figure 7 presents bar graphs demonstrating apoptosis level in DU145 cells treated by compounds 7, 9, 12 and 18 for 2 hours using annexin V-FITC apoptosis detection kit (BD Biosciences) according to the manufacturer's protocol and monitored via flow-cytometry. FITC: fluorescein isothiocyanate.

[040] Figure 8 presents a scheme showing a possible mechanism of hydrogen peroxide generation.

DETAILED DESCRIPTION OF THE INVENTION

[041 ] The present invention, in some embodiments thereof, relates to small molecules capable of selectively targeting of proteins that are overexpressed in cells (e.g., cancer cells). In some embodiments, the disclosed small molecules induce the formation of reactive oxygen species (ROS).

[042] In some embodiments, the disclosed molecules are for use for treating a disease or disorder as described herein.

[043] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

The compounds:

[044] In some embodiments, there is provided herein a compound having the general Formula I:

wherein each of R^-R ⁶ represents a substituent as described below in some embodiments.

[045] In some embodiments, OR ¹ is an electron-withdrawing group.

[046] Non-limiting exemplary electron- withdrawing groups are selected from: carboxy, acyl halide, amide, sulfonate, and nitro.

[047] In some embodiments, R ¹ is alkyl. In some embodiments, R ¹ is C1-C4 alkyl.

[048] In some embodiments, substituents R ² to R ⁶ , in each instance, is hydrogen. In some embodiments, substituents R ² to R ⁶ , in each instance is alkyl. In some embodiments, substituents R ² to R ⁶ , in each instance, is cycloalkyl. In some embodiments, substituents R ² to R ⁶ , in each instance is aryl. In some embodiments, substituents R ² to R ⁶ , in each instance, is heteroalicyclic. In some embodiments, substituents R ² to R ⁶ , in each instance, is heteroaryl. In some embodiments, substituents R ² to R ⁶ , in each instance, is alkoxy. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise hydroxyl. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise thiohydroxy. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise thioalkoxy. In some embodiments, substituents R ² to R ⁶ , in each instance comprise aryloxy. In some embodiments, substituents R ² to R ⁶ , in each instance comprise thioaryloxy. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise an amino group. In some embodiments, substituents R ² to R ⁶ , in each instance, is nitro. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise halo. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise trihalomethyl. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise cyano. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise amide. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise carboxy. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise sulfonyl. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise sulfoxy. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise sulfinyl. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise sulfonamide. In some embodiments, substituents R ² to R ⁶ , in each instance, comprise or is a fused ring.

[049] Herein, in some embodiments, by "substituents R ² to R ⁶ , in each instance" it may refer to one substituent from R ² to R ⁶ , two substituents from R ² to R ⁶ , three substituents from R ² to

R ⁶ , four substituents from R ² to R ⁶ , or five substituents from R ² to R ⁶ .

[050] In some embodiments, R ² is hydrogen.

[051] In some embodiments, R ¹ is methyl.

[052] In some embodiments, R ² is hydrogen.

[053] In some embodiments, at least one substituent selected from R ⁴ to R ⁵ is alkoxy. In some embodiments, both R ⁴ to R ⁵ are alkoxy. In some embodiments, either R ⁴ or R ⁵ is alkoxy. In some embodiments, the alkoxy is methoxy.

[054] In some embodiments, the disclosed compound is in the form of Formula IA (also referred to as: "compound 13"):

[055] In some embodiments, the disclosed compound is in the form of Formula IB (also referred to as "compound 12"):

[056] In some embodiments, the disclosed compound is in the form of Formula IC:

[057] As used herein, the term "alkyl" describes an aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 21 to 100 carbon atoms, and more preferably 21-50 carbon atoms. Whenever a numerical range; e.g., "21-100", is stated herein, it implies that the group, in this case the alkyl group, may contain 21 carbon atom, 22 carbon atoms, 23 carbon atoms, etc., up to and including 100 carbon atoms. In the context of the present invention, a "long alkyl" is an alkyl having at least 20 carbon atoms in its main chain (the longest path of continuous covalently attached atoms). A short alkyl therefore has 20 or less main-chain carbons. The alkyl can be substituted or unsubstituted, as defined herein.

[058] The term "alkyl", as used herein, also encompasses saturated or unsaturated hydrocarbon, hence this term further encompasses alkenyl and alkynyl.

[059] The term "alkenyl" describes an unsaturated alkyl, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond. The alkenyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.

[060] The term "alkynyl", as defined herein, is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.

[061] The term "cycloalkyl" describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system. The cycloalkyl group may be substituted or unsubstituted, as indicated herein.

[062] The term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi- electron system. The aryl group may be substituted or unsubstituted, as indicated herein.

[063] The term "alkoxy" describes both an -O-alkyl and an -O-cycloalkyl group, as defined herein.

[064] The term "aryloxy" describes an -O-aryl, as defined herein. [065] Each of the alkyl, cycloalkyl and aryl groups in the general formulas herein may be substituted by one or more substituents, whereby each substituent group can independently be, for example, halide, alkyl, alkoxy, cycloalkyl, alkoxy, nitro, amine, hydroxyl, thiol, thioalkoxy, thiohydroxy, carboxy, amide, aryl and aryloxy, depending on the substituted group and its position in the molecule. Additional substituents are also contemplated.

[066] The term "halide", "halogen" or "halo" describes fluorine, chlorine, bromine or iodine.

[067] The term "haloalkyl" describes an alkyl group as defined herein, further substituted by one or more halide(s).

[068] The term "haloalkoxy" describes an alkoxy group as defined herein, further substituted by one or more halide(s).

[069] The term "hydroxyl" or "hydroxy" describes a -OH group.

[070] The term "thiohydroxy" or "thiol" describes a -SH group.

[071] The term "thioalkoxy" describes both an -S-alkyl group, and a -S-cycloalkyl group, as defined herein.

[072] The term "thioaryloxy" describes both an -S-aryl and a -S-heteroaryl group, as defined herein.

[073] The term "amine" describes a - R'R" group, with R' and R" as described herein.

[074] The term "heteroaryl" describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.

[075] The term "heteroalicyclic" or "heterocyclyl" describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane, morpholino and the like.

[076] The term "carboxy" or "carboxylate" describes a -C(=0)-OR' group, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon) or heteroalicyclic (bonded through a ring carbon) as defined herein.

[077] The term "carbonyl" describes a -C(=0)-R' group, where R' is as defined hereinabove.

[078] The above-terms also encompass thio-derivatives thereof (thiocarboxy and thiocarbonyl). [079] The term "thiocarbonyl" describes a -C(=S)-R' group, where R' is as defined hereinabove.

[080] A "thiocarboxy" group describes a -C(=S)-OR' group, where R' is as defined herein.

[081] A "sulfinyl" group describes an -S(=0)-R' group, where R' is as defined herein.

[082] A "sulfonyl" or "sulfonate" group describes an -S(=0)2-R' group, where Rx is as defined herein.

[083] A "carbamyl" or "carbamate" group describes an -OC(=0)-NR'R" group, where R' is as defined herein and R" is as defined for R'.

[084] A "nitro" group refers to a -NO2 group.

[085] A "cyano" or "nitrile" group refers to a -C≡N group.

[086] As used herein, the term "azide" refers to a -N3 group.

[087] The term "sulfonamide" refers to a -S(=0) ₂ -NR'R" group, with R' and R" as defined herein.

[088] The term "phosphonyl" or "phosphonate" describes an -0-P(=0)(OR')2 group, with R' as defined hereinabove.

[089] The term "phosphinyl" describes a -PR'R" group, with R' and R" as defined hereinabove.

[090] The term "alkaryl" describes an alkyl, as defined herein, which substituted by an aryl, as described herein. An exemplary alkaryl is benzyl.

[091] The term "heteroaryl" describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or unsubstituted by one or more substituents, as described hereinabove. Representative examples are thiadiazole, pyridine, pyrrole, oxazole, indole, purine and the like.

[092] As used herein, the terms "halo" and "halide", which are referred to herein interchangeably, describe an atom of a halogen, that is fluorine, chlorine, bromine or iodine, also referred to herein as fluoride, chloride, bromide and iodide.

[093] The term "haloalkyl" describes an alkyl group as defined above, further substituted by one or more halide(s). Pharmaceutical Composition of the compounds:

[094] According to an aspect of embodiments of the invention there is provided a pharmaceutical composition comprising one or more compounds described herein and a pharmaceutically acceptable carrier.

[095] According to some embodiments of the invention, the composition is being packaged in a packaging material and identified in print, in or on the packaging material, for use in the treatment of a medical condition associated with any disease, medical condition, or disorder as described hereinthroughout.

[096] According to an aspect of embodiments of the invention there is provided a method of treating a medical condition associated with a disease, medical condition, or disorder as described hereinthroughout in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more compounds as described herein.

[097] The term "subject" (which is to be read to include "individual", "animal", "patient" or "mammal" where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on.

[098] In some embodiments, the subject is a human.

[099] According to an aspect of embodiments of the invention, there is provided a use of any one of the compounds described herein as a medicament, a drug, or a prodrug.

[0100] The compounds as described herein described hereinabove may be administered or otherwise utilized in this and in other aspects of the present invention, either as is, or as a pharmaceutically acceptable salt, solvate, hydrate or a prodrug thereof.

[0101] The phrase "pharmaceutically acceptable salt" refers to a charged species of the parent compound and its counter ion, which is typically used to modify the solubility characteristics of the parent compound and/or to reduce any significant irritation to an organism by the parent compound, while not abrogating the biological activity and properties of the administered compound. The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention. The phrase "pharmaceutically acceptable salts" is further meant to encompass salts of the active compounds (e.g., compounds as described herein) which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.

[0102] Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et ah, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compound as described herein to be converted into either base or acid addition salts.

[0103] The neutral forms of the compounds as described herein may be regenerated by contacting the salt with a base or acid and isolating the parent compounds in a conventional manner. The parent form of the compounds may differ from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the disclosed compound(s) for the purposes of the present invention.

[0104] The term "prodrug" refers to an agent, which is converted into the active compound (the active parent drug) in vivo. Prodrugs are typically useful for facilitating the administration of the parent drug. The prodrug may also have improved solubility as compared with the parent drug in pharmaceutical compositions. Prodrugs are also often used to achieve a sustained release of the active compound in vivo.

[0105] The compounds described herein may possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are encompassed within the scope of the present invention.

[0106] As used herein, the term "enantiomer" describes a stereoisomer of a compound that is superposable with respect to its counterpart only by a complete inversion/reflection (mirror image) of each other. Enantiomers have "handedness" since they refer to each other like the right and left hand. Enantiomers have identical chemical and physical properties except when present in an environment which by itself has handedness, such as all living systems.

[0107] The compounds described herein may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0108] The term "solvate" refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a solute (the conjugate described herein) and a solvent, whereby the solvent does not interfere with the biological activity of the solute. Suitable solvents include, for example, ethanol, acetic acid and the like. The term "hydrate" refers to a solvate, as defined hereinabove, where the solvent is water.

[0109] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the compounds described herein (as active ingredient), or physiologically acceptable salts or prodrugs thereof, with other chemical components including, but not limited to, physiologically suitable carriers, stabilizers, preservatives, chelating agents, viscosity modifying agents, excipients, lubricants, buffering agents, antibacterial agents, bulking agents (e.g., mannitol), antioxidants (e.g. , ascorbic acid or sodium bisulfite), anti-inflammatory agents, anti-viral agents, chemotherapeutic agents, anti-histamines and the like. The purpose of a pharmaceutical composition is to facilitate administration of a compound to a subject. The term "active ingredient" refers to a compound e.g., a compound disclosed herein, which is accountable for a biological effect.

[0110] The term "pharmaceutically acceptable carrier" refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

[0111] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a drug. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0112] Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences" Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference. [0113] Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. The dosage, as described and specified herein, may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. l).

[0114] The pharmaceutical composition may be formulated for administration in either one or more of routes depending on whether local or systemic treatment or administration is of choice, and on the area to be treated. As further described hereinthroughout, administration may be done orally, by inhalation, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, intramuscular or intravenous injection, or topically (including ophtalmically, vaginally, rectally, intranasally).

[0115] Formulations for topical administration may include but are not limited to lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0116] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, pills, caplets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable.

[0117] Formulations for parenteral administration may include, but are not limited to, sterile solutions which may also contain buffers, diluents and other suitable additives. Slow release compositions are envisaged for treatment.

[0118] The amount of a composition or a compound to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0119] The pharmaceutical composition may further comprise additional pharmaceutically active or inactive agents such as, but not limited to, an antibacterial agent, an antioxidant, a buffering agent, a bulking agent, a surfactant, an anti-inflammatory agent, an anti- viral agent, a chemotherapeutic agent and an anti-histamine.

[0120] Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.

Methods of treatments:

[0121] In some embodiments, the present invention provides a method comprising: identifying a subject having overexpression of protease (e.g., cysteine protease)-associated disease and administering to the subject an effective amount of a compound represented by the structure of formula I or any embodiment thereof.

[0122] The term "cysteine protease" or "cysteine peptidase" refers to the family of peptidases which have a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad. The first step is deprotonation of a thiol in the enzyme's active site by an adjacent amino acid with a basic side chain, usually a histidine residue. Cysteine proteases may have characteristic molecular topologies, which can be seen not only in their three-dimensional structures, but also in the two-dimensional structures. Cysteine proteases are divided into clans (proteins which are evolutionary related), and further sub-divided into families, on the basis of the architecture of their catalytic dyad or triad. The cysteine proteases family includes, but not limited to, papain, cathepsins, caspases, legumain, calpains, calpain and adenain.

[0123] In some embodiments, the term "cysteine protease" refers to deubiquitinating enzymes.

[0124] Deubiquitinating enzymes (DUBs), also known as "deubiquitinating peptidases", "deubiquitinating isopeptidases", "deubiquitinases", "ubiquitin proteases", "ubiquitin hydrolases", "ubiquitin isopeptidases", are a large group of proteases that cleave ubiquitin from proteins and other molecules.

[0125] Families of DUBs may be selected from, without being limited thereto, the ubiquitin- specific protease (USP/UBP) superfamily, the ovarian tumor (OTU) superfamily (OTUB 1, OTUB2); the Machado-Josephin domain (MJD) superfamily; and (ATXN3, ATXN3L) the ubiquitin C-terminal hydrolase (UCH) superfamily (e.g., BAP1, UCHL1, UCHL3, UCHL5).

[0126] Non-limiting examples of the ubiquitin- specific protease (USP/UBP) superfamily are USP1, USP2, USP3, USP4, USP5, USP6, USP7, USP8, USP9X, USP9Y, USP10, USP11, USP12, USP13, USP14, USP15, USP16, USP17, USP17L2, USP17L3, USP17L4, USP17L5, USP17L7, USP17L8, USP18, USP19, USP20, USP21, USP22, USP23, USP24, USP25, USP26, USP27X, USP28, USP29, USP30, USP31, USP32, USP33, USP34, USP35, USP36, USP37, USP38, USP39, USP40, USP41, USP42, USP43, USP44, USP45, and USP46.

[0127] In some embodiments, the ubiquitin-specific protease is USP2. USP2 is associated with aggressive prostate cancer and triple negative breast cancer. USP2 is further associated with various known substrates in cell and affects the pathways that these substrates are involved in. The best-characterized substrate of USP2 is fatty acid synthase (FAS), responsible for protection of prostate cancer cells from apoptosis.

[0128] Certain embodiments of the present invention provide methods for treating disease or disorder is associated with overexpression of cysteine protease. Certain embodiments of the present invention provide methods for treating disease or disorder is associated with protease.

[0129] Certain embodiments of the present invention provide methods for the treatment, prevention, or amelioration of diseases, disorders, and conditions associated with overexpression of cysteine protease in an individual in need thereof. In another embodiment, the method comprises the step of contacting the cell with a composition comprising one or more compounds disclosed herein.

[0130] Also contemplated are methods for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with overexpression of cysteine protease. Overexpression of cysteine protease associated diseases, disorders, and conditions may include, without being limited thereto, inflammatory diseases, neurodegenerative diseases, cancer, and viral diseases. In certain embodiments, the neurodegenerative disease may be ALS or FTD. In certain embodiments, the neurodegenerative disease may be familial or sporadic.

[0131] The terms "cancer" and "tumor" are used interchangeably herein to describe a class of diseases in which a group of cells display uncontrolled growth (division beyond the normal limits). The term "cancer" encompasses malignant tumor as well as disease conditions evolving from primary or secondary tumors. The term "malignant tumor" describes a tumor which is not self-limited in its growth, is capable of invading into adjacent tissues, and may be capable of spreading to distant tissues (metastasizing). The term "primary tumor" describes a tumor that is at the original site where it first arose. The term "secondary tumor" describes a tumor that has spread from its original (primary) site of growth to another site, close to or distant from the primary site.

[0132] In some embodiments, the cancer is be selected from: carcinoma, sarcoma, leukemia, lymphoma, and germinoma. [0133] Non-limiting examples of cancers which can be treated according to some embodiments of the invention can be solid or non- solid cancer and/or cancer metastasis, including, without being limited thereto, adrenocortical carcinoma, hereditary; bladder cancer; breast cancer; breast cancer, ductal; breast cancer, invasive intraductal; breast cancer, sporadic; breast cancer, susceptibility to; breast cancer, type 4; breast cancer, type 4; breast cancer- 1; breast cancer-3; breast-ovarian cancer; Burkitt's lymphoma; cervical carcinoma; colorectal adenoma; colorectal cancer; colorectal cancer, hereditary nonpolyposis, type 1; colorectal cancer, hereditary nonpolyposis, type 2; colorectal cancer, hereditary nonpolyposis, type 3; colorectal cancer, hereditary nonpolyposis, type 6; colorectal cancer, hereditary nonpolyposis, type 7; dermatofibro sarcoma protuberans; endometrial carcinoma; esophageal cancer; gastric cancer, fibrosarcoma, glioblastoma multiforme; glomus tumors, multiple; hepatoblastoma; hepatocellular cancer; hepatocellular carcinoma; leukemia, acute lymphoblastic; leukemia, acute myeloid; leukemia, acute myeloid, with eosinophilia; leukemia, acute nonlymphocytic; leukemia, chronic myeloid; Li-Fraumeni syndrome; liposarcoma, lung cancer; lung cancer, small cell; lymphoma, non-Hodgkin's; lynch cancer family syndrome II; male germ cell tumor; mast cell leukemia; medullary thyroid; medulloblastoma; melanoma, meningioma; multiple endocrine neoplasia; myeloid malignancy, predisposition to; myxosarcoma, neuroblastoma; osteosarcoma; ovarian cancer; ovarian cancer, serous; ovarian carcinoma; ovarian sex cord tumors; pancreatic cancer; pancreatic endocrine tumors; paraganglioma, familial nonchromaffin; pilomatricoma; pituitary tumor, invasive; and prostate adenocarcinoma; prostate cancer; renal cell carcinoma, papillary, familial and sporadic; retinoblastoma; rhabdoid predisposition syndrome, familial; rhabdoid tumors; rhabdomyosarcoma; small-cell cancer of lung; soft tissue sarcoma, squamous cell carcinoma, head and neck; T-cell acute lymphoblastic leukemia; Turcot syndrome with glioblastoma; tylosis with esophageal cancer; uterine cervix carcinoma, Wilms' tumor, type 2; and Wilms' tumor, type 1, and the like.

[0134] According to one embodiment of this aspect of the present invention, the cancer is colonorectal cancer.

[0135] As used herein, the phrase "colorectal cancer" refers to malignant tumors of the epithelium of the colon or rectal, including but not limited to squamous cell (epidermoid) carcinomas, cloacogenic (basaloid transitional cell) tumors, and adenocarcinomas.

[0136] The following are non-limiting examples of the solid cancers treatable with the compounds described herein: ovarian, pancreas, brain (e.g., glioblastoma), colon, rectal, colorectal, melanoma, lung, breast (e.g., breast adenocarcinoma), kidney (renal), and prostate cancers. [0137] In some embodiments cancers treatable with the compounds described herein are non- solid cancers e.g., leukemia (e.g., T cell leukemia).

[0138] The term "cancer metastases", or any grammatical derivative thereof, describes cancer cells which have "broken away", "leaked", or "spilled" from a primary tumor, entered the lymphatic and/or blood vessels, circulated through the lymphatic system and/or bloodstream, settled down and proliferated within normal tissues elsewhere in the body thereby creating a secondary tumor.

[0139] In another embodiment, the disease is associated with overly active nuclear factor kappa B (NF-κΒ). Therefore, in another embodiment, there is provided herein a method of inhibiting a nuclear factor kappa B (NF-κΒ) pathway in a cell, comprising the step of contacting the cell with a composition comprising one or more compounds disclosed herein.

The Synthesis

Synthesis of C5-C '-substituted 1,2 -naphthoquinones

[0140] In some embodiments, there is provided a synthesis route for the preparation of a compound of the Formula II:

In some embodiments, the synthesis route comprises:

b) contactin a compound of Formula III:

with hyper-valent iodine reagent in an organic solvent to give Formula IV: followed by reaction with R^OH and a metal chloride, to give Formula II.

[0141] In some embodiments, R ¹ is C1-C4 alkyl. In some embodiments, substituents R ³ to R ⁶ , in each instance, comprise or are selected from: hydrogen, alkyl, cycloalkyl, aryl, heteroalicyclic, heteroaryl, alkoxy, hydroxy, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amide, carboxy, sulfonyl, sulfoxy, sulfinyl, or sulfonamide, and wherein at least one substituent selected from R ³ to R ⁶ is alkoxy.

[0142] In some embodiments, R ¹ is methyl, and R ⁶ is hydrogen. In some embodiments, R ³ is hydrogen. In some embodiments, either R ⁴ or R ⁵ is alkoxy. In some embodiments, the alkoxy is methoxy.

[0143] In some embodiments, the reaction of step (a) is conducted at a temperature range of about 10°C to about 90°C. In exemplary embodiments, the reaction is performed at elevated temperature.

[0144] In some embodiments, the reaction of step (a) is conducted for at least 30 min, 1 h , 2 h, 3 h, 4 h , 5 h, 6 h, 7 h , 8 h, 9 h, 10 h , 11 h, 12 h, 13 h , 14 h, or 15 h, including any value and range therebetween. In some embodiments, the reaction of step (a) is conducted while stirring. In exemplary embodiments, the reaction of step (a) is conducted for 12 h to 14 h.

[0145] In some embodiments, the organic solvent is dimethyl sulfoxide (DMSO).

[0146] The term "hyper-valent iodine compound" refers to three-, five-, seven-, or eight- coordinate iodine compounds.

[0147] In some embodiments, the hyper-valent iodine reagent is selected from, without being limited thereto, phenyliodine diacetate (PIDA), [bis(trifluoroacetoxy)iodo]benzene (PIFA), Dess-Martin periodinane (DMP), and 2-iodoxybenzoic acid (IBX).

[0148] In some embodiments, the hyper-valent iodine reagent and the compound of the Formula III are in molar ratio of from 1: 3.5 to 1:4.5, respectively. In some embodiments, the molar ratio is about 1:4.

[0149] In some embodiments, step (b) is performed in the presence of an oxidizing agent.

[0150] In some embodiments, the oxidizing agent is iodate salt. [0151] Non-limiting exemplary iodate salt is selected from: calcium iodate, sodium iodate, potassium iodate, magnesium iodate, zinc iodate, cupric iodate, and manganese iodate.

[0152] In exemplary embodiments, the oxidizing agent is sodium iodate.

[0153] In some embodiments, the contacting of step (b) is performed in the presence of metal ion. In some embodiments, the metal ion is obtained from metal chloride source. Non-limiting examples of metal ion are cerium (Ce ³⁺ ), lanthanum (La ³⁺ ), copper (Cu ²⁺ ), and nickel (Ni ²⁺ ).

[0154] In some embodiments, steps (a) and (b) are conducted in one pot. In some embodiments, steps (a) and (b) are conducted simultaneously. In some embodiments, steps (a) and (b) are conducted sequentially.

[0155] In some embodiments, steps (a) and (b) are followed by step (c) of purification of the product, by any method known in the art (e.g., by using a column).

[0156] Reference is now made to Figure 3A which presents a scheme showing non-limiting exemplary synthesis of C5-C7-substituted 1,2-naphthoquinones according to the disclosed synthesis route.

[0157] In exemplary embodiments, a reaction of 5-, 6- and 7- methoxy and/or 6-OTs tetralones is performed. "Ts" may refer to a tosyl group.

[0158] In exemplary embodiments, the reaction affords the corresponding 5-, 6- and 7- substituted 1,2 naphthoquinones (compound 8-11, respectively; Figure 3A).

[0159] In exemplary embodiments, reaction of compounds 9-11 with CeCh ^fhO and sodium iodate in MeOH affords obtaining compounds 12-14 (Figure 3A).

C4-substituted 1,2-naphthoquinones:

[0160] In some embodiments, the synthesis comprises contacting methyl 3- mercaptopropionate (MMP), 3- mercaptopropionic acid (MPA), or 2-(Boc-amino) ethanethiol with 1,2 naphthoquinone, or a derivative thereof, so as to obtain the corresponding compounds 2-4 as described herein, or a derivative thereof.

[0161] In exemplary embodiments, methyl 3 -mercaptopropionate (MMP), 3- mercaptopropionic acid (MPA), or 2-(Boc-amino) ethanethiol are reacted with 1,2 naphthoquinone at room temperature (e.g., 15-30 °C), and may be further allowed to stir for e.g., 30 min., 1 h, 2, h or 3h (including any value therebetween) at elevated temperature.

[0162] In some embodiments, "elevated temperature" refers to e.g., 35 °C, 40 °C, 50 °C, 60 °C, 70 °C, or 80°C, including any value therebetween.

[0163] In exemplary embodiments, 4-methoxy 1,2-naphthoquinone (compound 7), or a derivative thereof, is obtained by the treatment of 1,2 naphthoquinone, or a derivative thereof, with methanol in the presence of cerium ions (e.g., in a salt CeCb). In some embodiments, 4- methoxy 1,2-naphthoquinone (compound 7), or a derivative thereof, is obtained by the treatment of 1,2 naphthoquinone with methanol in the presence of cerium ions (e.g., in a salt CeCl ₃ ) and further in the presence of sodium iodate.

General

[0164] As used herein the term "about" refers to ± 10 %.

[0165] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". The term "consisting of means "including and limited to". The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0166] The word "exemplary" is used herein to mean "serving as an example, instance or illustration". Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.

[0167] The word "optionally" is used herein to mean "is provided in some embodiments and not provided in other embodiments". Any particular embodiment of the invention may include a plurality of "optional" features unless such features conflict.

[0168] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0169] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0170] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0171] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0172] As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0173] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B " or "A and B."

[0174] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0175] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0176] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0177] A collection of quinone-containing molecules was tested for USP2 inhibition (e.g., compounds listed in Figure 1). The inhibiting mechanism DUBs through ROS -induced damage to the enzyme was investigated.

[0178] The investigations started with a comparison between the non-substituted ortho- naphthoquinone 1 and various /?ara-naphthoquinone, compounds 15-17 and the anticancer drugs (compounds 22-24). The apparent superior inhibitory effect of compound 1 relative to these six compounds triggered efforts towards the synthesis of substituted 1,2- naphthoquinones, of which three compounds (25, 12, and 7) were identified to be more potent USP2 inhibitors than β-lapachone.

[0179] In the search for the origin of the superiority of ortho- vs. /?ara-naphthoquinones, both the reduction potentials (quinone/semiquinone radical, determined under anaerobic conditions) and the electro-catalytic activity for reduction of oxygen (to 0 ₂ ^" , which undergoes spontaneous disproportionation to H2O2 and O2) were determined for 11 derivatives.

EXAMPLE 1

SYNTHESIS AND CHEMICAL CHARACTERIZATION

Synthesis of C4-substituted 1,2-naphthoquinones (compounds 2-7):

[0180] In brief, to a stirred suspension of compound 1 (250 mg, 1.58 mmol) and K2CO3 (1.30 g, 9.49 mmol) in DMF (10 mL), methyl 3-mercaptopropionate (175 μΐ, 1.58 mmol) was added at room temperature and allowed to stir for 3h at 50 °C.

[0181] In exemplary procedures, upon the facile Michael addition with the methyl 3- mercaptopropionate (MMP), 3- mercaptopropionic acid (MPA), or 2-(Boc-amino) ethanethiol to the commercially available 1,2 naphthoquinone (compound 1 in Figure 1), the compounds 2-4 (in Figure 1) were obtained, respectively, appended with the acid or the amine functionality suitable for the further functionalization (scheme in Figure 2A). Direct incorporation of the amine onto the C4 position was achieved via the reaction of sodium azide under acidic conditions, leading to compound 5. 4-methoxy 1,2-naphthoquinone (compound 7 in Figure 1) was obtained by the treatment of 1,2 naphthoquinone with methanol in the presence of equimolar CeCl ₃ '7H20 and sodium iodate. Compound 6 in Figure 1 is commercially available and was purchased from Acros chemicals. Synthesis of C5-C '-substituted 1, 2 -naphthoquinones :

[0182] In brief, reaction of 5-, 6- and 7- methoxy and 6-OTs tetralones with 2-iodoxybenzoic acid (IBX) in DMSO at 80 °C afforded the corresponding 5-, 6- and 7-substituted 1,2 naphthoquinones (compounds 8-11, respectively; Figure 2A). Reaction of compounds 9-11 with CeCl3.7H ₂ 0 and sodium iodate in MeOH afforded the compounds 12-14 (Figure 3A).

[0183] In exemplary procedures, 5-methoxy tetralone (100 mg, 0.567 mmol) was dissolved in DMSO (10 mL) and IBX (635 mg, 2.27mmol) was added and heated at 80 °C for 10-12 hr until the TLC showed the complete disappearance of the starting material. The reaction mixture was then quenched with water and extracted with EtOAc and the combined organic layers were washed with saturated sodium carbonate solution and purified by column using Hexanes- EtOAc as eluents to obtain compound 8 as a red solid (69 mg, 65% yield).

[0184] Compound 9 (80 mg, 75% yield) was prepared starting from 6-methoxy tetralone (100 mg, 0.567 mmol) according to the procedure described above to give the desired product as a red solid.

[0185] Compound 10 (73 mg, 68% yield) was prepared starting from 7-methoxy tetralone (100 mg, 0.567 mmol) according to the procedure described above to give the desired product as a red solid.

General procedure for the synthesis of di-suhstituted 1,2 naphthoquinones :

[0186] Compound 12: 6-methoxy tetralone (100 mg, 0.567 mmol) was dissolved in DMSO (10 mL) and IBX (635 mg, 2.27 mmol) was added followed by heating at 80 °C for 10-12 h until the TLC showed the complete disappearance of the starting material. The reaction mixture was then quenched with water and extracted with EtOAc and the combined organic layers were washed with saturated sodium carbonate solution. The crude material was then dissolved in 5 ml of MeOH and NaI0 ₃ (112 mg, 0.567 mmol) and CeCl ₃ 7H ₂ 0 (470 mg, 0.567 mmol) were added in one portion and stirred vigorously at room temperature. After 20-30 minutes, the solvent was evaporated under reduced pressure followed by the addition of water and EtOAc. The crude material was purified using CHCb/EtOAc as eluents to give compound 12 as a yellow solid (43 mg, 35% yield over two steps).

[0187] Similarly, compound 13 (38 mg, 31% yield over two steps) was prepared starting from 7-methoxy tetralone (100 mg, 0.567 mmol ) according to the procedure described above which was obtained as a red solid.

[0188] Similarly, compound 14 (43 mg, 38% yield over two steps) was prepared starting from 6-OTosyl tetralone (100 mg, 0.316 mmol) according to the procedure described above as yellow solid. Synthesis of 3-hydroxy lapachone (compound 25):

[0189] Compound 25 was prepared according to the literature procedure (J. S. Sun, A. H. Geiser and B. Frydman, Tetrahedron Lett., 1998, 39, 8221-8224).

Characterization :

[0190] General methods' ^X H and ¹³ C NMR spectra were recorded using CDC13 and DMSO-d6 as solvents. Chemical shifts were reported in δ units (ppm) with reference to TMS as an internal standard, and J values are given in Hz. ^l H and ¹³ C-NMR spectra were recorded on a Bruker AMX-400 MHz spectrometer. Mass determination of the materials was carried out using an LCQ Fleet Ion Trap (Thermo Scientific). Flash column chromatography was carried out with silica gel (220-440 mesh). The reactions were carried out in oven-dried glassware under nitrogen. Chemicals and compounds 1, 6, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 were purchased from Aldrich, Fluka and Alfa Aesar. Commercial reagents were used without further purification. Analytical thin-layer chromatography (TLC) was performed on pre-coated plates (0.25 mm, silica gel 60 F254). Compound spots were visualized by UV light (254 nm).

[0191] TLC showed the complete disappearance of starting material. The reaction was quenched with water, extracted with EtOAc and purified by column using CHC13-EtOAc as eluents to give compound 2, as a dark red solid (312mg, 52% yield). 1H NMR (CDC13) δ 8.09 (dd, J = 7.6, 1.2 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.61 (td, J = 7.7, 1.3 Hz, 1H), 7.51 (t, J = 7.5 Hz, 1H), 6.35 (s, 1H), 3.69 (s, 3H), 3.26 (t, J = 7.1 Hz, 2H), 2.77 (t, J = 7.1 Hz, 2H); 13C NMR (CDC13) δ 179.45, 176.37, 171.15, 158.89, 135.19, 133.55, 131.42, 130.58, 129.50, 125.31, 119.76, 52.40, 32.28, 26.45. HRMS (ESI) exact mass calcd. for Ci ₇ H ₉ 0 ₄ [M+H]+ 277.0501, found [M+H]+ 277.0501.

[0192] Compound 3 (152 mg, 37% yield) was prepared starting from compound 1 (250 mg, 1.58 mmol) according to the procedure described above, which was isolated as a red solid. 1H NMR (DMSO) δ 12.85- 12.35 (bs, 1H), 8.05 - 7.97 (m, 1H), 7.85 - 7.74 (m, 2H), 7.66 (td, J = 7.4, 1.4 Hz, 1H), 6.45 (s, 1H), 3.34 (t, J = 6.8 Hz, 2H), 2.75 (t, J = 6.8 Hz, 2H); ¹³ C NMR (DMSO) 6 178.71, 175.74, 172.30, 156.73, 135.07, 133.09, 131.15, 130.57, 128.31, 124.92, 120.16, 32.10, 25.99. High resolution mass spectra (HRMS) electrospray ionization (ESI) exact mass calcd. for Ci ₃ Hi ₀ O ₄ SNa [M+Na]+ 285.0198, found [M+H]+ 285.0138.

[0193] Compound 4 (280 mg, 53% yield) was prepared starting from compound 1 (250 mg, 1.58 mmol) according to the procedure described above, which was isolated as a pale yellow solid. ^l H NMR (CDCB) δ 8.16 (dd, J = 7.6, 1.2 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.68 (td, J = 7.7, 1.3 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 6.46 (s, 1H), 4.96 (s, 1H), 3.53 (dd, J = 12.5, 6.2 Hz, 2H), 3.23 (t, J = 6.5 Hz, 2H), 1.46 (s, 9H).13C NMR (CDC13) δ 179.39, 176.27, 158.97, 155.74, 135.05, 133.55, 131.25, 130.43, 129.33, 125.27, 119.82, 80.10, 38.55, 31.79, 28.37(3C). HRMS (ESI) exact mass calcd. for CioH ₈ N0 ₂ [M+H]+ 334.1113, found [M+H]+ 334.1102.

[0194] Compound 5 (480 mg, 88% yield) was prepared starting from compound 1 (500 mg, 3.16 mmol) according to the literature procedure, which was isolated as red solid.1 ^l H NMR (DMSO) δ 8.4-8.25 (bs, 1H), 8.22-8.09 (s, 1H), 8.04 (d, J = 7.7 Hz, 1H), 7.97 (dd, J = 7.6, 1.2 Hz, 1H), 7.81 (td, J = 7.7, 1.4 Hz, 1H), 7.69 (td, J = 7.5, 0.7 Hz, 1H), 5.73 (s, 1H); 13C NMR (DMSO) 5 182.16, 174.62, 157.96, 134.21, 131.61, 131.59, 130.45, 127.73, 123.94, 100.99. HRMS (ESI) exact mass calcd. for OoH ₈ N0 ₂ [M+H]+ 174.0555, found [M+H]+ 174.0505.

[0195] Figure 2B present H-NMR and C-NMR spectra of compounds 1-3, and 5-7.

[0196] Compound 8 ^l U NMR (CDC1 ₃ ) δ 7.96 (d, J = 10.4 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 6.34 (d, J = 10.4 Hz, 1H), 3.94 (s, 3H). ¹³ C NMR (CDCI3) δ 181.14, 179.55, 156.82, 139.44, 132.97, 132.34, 126.17, 123.20, 122.43, 118.24, 56.34. HRMS (ESI) exact mass calcd. for C11H9O3 [M+H] ⁺ 189.0552, found [M+H] ⁺ 189.0550.

[0197] Compound 9 ^l U NMR (CDCI3) δ 8.09 (d, J = 8.6 Hz, 1H), 7.35 (d, J = 10.1 Hz, 1H), 6.93 (dd, J = 8.6, 2.4 Hz, 1H), 6.82 (d, J = 2.3 Hz, 1H), 6.41 (d, J = 10.1 Hz, 1H), 3.92 (s, 3H). ¹³ C NMR (CDC1 ₃ ) 6 181.67, 177.43, 165.84, 144.93, 137.07, 133.37, 128.76, 125.15, 1 16.09, 114.90, 56.07. HRMS (ESI) exact mass calcd. for C11H9O3 [M+H] ⁺ 189.0552, found [M+H] ⁺ 189.0578.

[0198] Compound 10 ^l H NMR (CDCI3) δ 7.58 (d, J = 2.7 Hz, 1H), 7.36 (d, J = 10.1 Hz, 1H), 7.24 (d, J = 1.1 Hz, 1H), 7.10 (dd, 7 = 8.4, 2.7 Hz, 1H), 6.26 (d, J = 10.1 Hz, 1H), 3.88 (s, 3H). ¹³ C NMR (CDC1 ₃ ) 5 181.09, 179.12, 162.00, 145.70, 133.29, 131.70, 128.05, 125.28, 121.81, 114.87, 56.03. HRMS (ESI) exact mass calcd. for C11H9O3 [M+H] ⁺ 189.0552, found [M+H] ⁺ 189.0550.

[0199] Compound 12 ^l U NMR (CDCI3) δ 8.07 (d, J = 8.6 Hz, 1H), 7.30 (d, J = 2.5 Hz, 1H), 7.00 (dd, J = 8.6, 2.5 Hz, 1H), 5.93 (s, 1H), 3.99 (s, 3H), 3.93 (s, 3H). ¹³ C NMR (CDCI3) δ 180.24, 178.12, 168.05, 165.25, 134.40, 132.06, 123.92, 116.24, 110.64, 103.55, 56.87, 56.05. HRMS (ESI) exact mass calcd. for Ci ₂ Hn0 ₄ [M+H] ⁺ 219.0657, found [M+H] ⁺ 219.0629.

[0200] Compound 13 ^l H NMR (CDCI3) δ 7.76 (d, J = 8.7 Hz, 1H), 7.60 (d, J = 2.7 Hz, 1H), 7.16 (dd, J = 8.7, 2.8 Hz, 1H), 5.87 (s, 1H), 4.00 (s, 3H), 3.91 (s, 3H). ¹³ C NMR (CDCI3) δ 179.80, 179.76, 169.60, 162.47, 132.20, 126.74, 124.88, 121.34, 113.22, 101.29, 56.86, 56.02. HRMS (ESI) exact mass calcd. for Ci ₂ Hn0 ₄ [M+H] ⁺ 219.0657, found [M+H] ⁺ 219.0617.

[0201] Compound 14 ^l H NMR (CDCI3) δ 8.02 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.66 (d, J = 2.3 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.04 (dd, J = 8.4, 2.3 Hz, 1H), 5.99 (s, 1H), 4.01 (s, 3H), 2.47 (s, 3H); ¹³ C NMR (CDCb) δ 179.01, 178.28, 167.22, 154.60, 146.35, 134.35, 132.06, 131.16, 130.24 (2C), 128.83, 128.63 (2C), 124.80, 119.38, 104.05, 57.15, 21.92. HRMS (ESI) exact mass calcd. for Ci ₈ Hi ₅ 0 ₆ S [M+H] ⁺ 359.0589, found [M+H] ⁺ 359.0549.

[0202] Compound 25 ^l U NMR (CDCb) δ 8.04 (dd, 7 = 7.6, 1.0 Hz, 1H), 7.83 (d, 7 = 7.3 Hz, 1H), 7.65 (td, 7 = 7.7, 1.3 Hz, 1H), 7.51 (td, 7 = 7.6, 1.0 Hz, 1H), 3.93 (t, 7 = 5.1 Hz, 1H), 2.81 (dd, 7 = 17.7, 4.9 Hz, 1H), 2.62 (dd, 7 = 17.7, 5.3 Hz, 1H), 2.15 (d, 7 = 9.5 Hz, 1H), 1.51 (s, 3H), 1.45 (s, 3H). ¹³ C NMR (CDCb) δ 179.66, 178.88, 161.67, 135.02, 132.22, 131.07, 130.20, 128.88, 124.51, 110.56, 81.62, 68.42, 25.52, 25.23, 22.23. HRMS (ESI) exact mass calcd. for Ci ₅ Hi ₅ 0 ₄ [M+H] ⁺ 259.0970, found [M+H] ⁺ 259.0954.

[0203] Figure 3B presents HNMR spectra of compounds 8-13.

EXAMPLE 2

USP2 INHIBITIONS

[0204] A focused collection of quinone-containing molecules (compounds 15-24, summarized in Figure 4) was obtained from commercial sources, which together with all the synthesized quinone derivatives described above, were tested for USP2 inhibition using a quenching pair assay. Among the non-substituted derivatives, o/ /zo-naphthoquinone, compound 1, exhibited full inhibition at 5 μΜ, while only 20% inhibition was obtained for the /?ara-naphthoquinones counterpart compound 15 at the same concentration. Adding hydroxyl substituents, to give compound 16 or /?ara-quinone, to give compound 17 led to marginal improvements relative to the original compound 15.

[0205] The comparison between β-lapachone (compound 18) and dehydro-a-lapachone (19) revealed complete inhibition against USP2 at 5 μΜ for both, however at 1 μΜ β-lapachone displayed 100% inhibition whereas the activity of dehydro-a-lapachone dropped to 11%. A similar comparison with the ηοΓ-β-lapachone (20) and nor-a-lapachone derivatives (compound 21) disclosed 100% and 68% USP2 inhibition, respectively. Taken together, these results show that 1,2-quinones are consistently more potent USP2 inhibitors than 1,4-quinones.

[0206] Armed with these new findings, some selected anticancer drugs [doxorubicin (compound 22), mytomycin C (compound 23) and menadione (compound 24)] were screened as to establish if DUBs are possible targets for them. These examinations revealed that compounds 22-24 did not exhibited appreciable inhibition against USP2, even at 5 μΜ concentrations.

[0207] Compounds 2-7 contain different substitution on C4 position of the ortho- naphthoquinone 1: S-alkyl groups in compounds 2-4, amine in compound 5, SO3 ^" in compound 6, and methoxy in compound 7. Compounds 2-4 did not exhibit measurable activity against USP2 at 1 μΜ, which might be attributed to oxidation of the sulfide-moiety therein by the ROS. Compound 6 with its electron-withdrawing sulfonyl group did not show any inhibition at 1 μΜ, while compounds 5 and 7 with their electron-donating groups (-NH ₂ and -OCH3, respectively) exhibited substantially increased activity relative to the parent compound 1. Here 33% inhibition was observed at 500 nm for compound 5 and nearly complete inhibitory activity at 400 nM for compound 7. Taken together, the methoxy substitution in compound 7 led to an about 12-fold increase in the activity compared to the unsubstituted naphthoquinone (compound 1), which indicates that electron-donating groups provide a beneficial effect when presented on C4.

[0208] Compounds in which a methoxy group is present on the non-quinonic ring of 1,2 naphthoquinones, at positions 5, 6 and 7 (compounds 8-10) were also prepared, however none of them displayed improved inhibitory activity at 1 μΜ. In contrast, compounds 12-14 which have C5- or C6-substituents in addition to the C4-OCH ₃ , were potent inhibitors. In these cases, 47% inhibition was observed at 300 nM for compound 12, 28% at 300 nM for compound 13, and 32% inhibition at 500 nM compound 14. 3-hydroxy β-lapachone (compound 25, Figure 4), exhibited 78% inhibition at 300 nm, the best candidate in the tricyclic class of compounds.

[0209] Having identified compound 12 as the most potent bicyclic inhibitor, its ^i _ac t was determined and found to be 3333 M ^_1 S ^_1 (Figure 5A). To verify that compound 12 also inhibit USP2 via the oxidation mechanism proposed for β-lapachone, the mass of the enzyme was measured before and after treatment with compound 12.

[0210] Figure 5B presents the mass spectrometry of USP2 treated with DMSO with compound 12 for 15 min.

[0211] The 32 Da increase is in agreement with the conversion of the catalytic Cys to sulfinic acid as illustrated in Figure 5A, Figure 5B, and the schematic illustration in Figure 6 demonstrating that the mechanism of inhibition by β-lapachone proceeds via ROS generation and irreversible oxidation of the catalytic Cys to the sulfinic acid form.

EXAMPLE 3

CELL STUDY

[0212] Cell culture: In exemplary procedures, DU145 cells were grown in EMEM medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin in 37 °C humidified incubator with a 5% C0 ₂ , 95% air atmosphere.

[0213] Apoptosis studies: Induction of apoptosis in DU-145 cell line by treatment with compounds 7, 9, 12 and 18 were determined after 2 h incubation in a dose dependent manner, using annexin V-FITC apoptosis detection kit (BD Biosciences) according to the manufacturer's protocol and monitored via flow-cytometry (fluorescence-activated cell sorting, FACS). Briefly, 2 x 10 ⁵ cells/well were seeded in 6-well plates and treated with inhibitor for 2 hr in a dose dependent manner. The cells were then harvested and washed with PBS. Next, the cells were re-suspended with 85 μΐ _^ binding buffer and stained with 10 μΐ _^ annexin V-FITC reagent and 5 μΐ _^ propidium iodide (PI) for 15 min in the dark. The increase in fluorescence, which indicates the apoptosis level in the treated cells, were monitored using flow cytometry and compared to untreated cells containing DMSO as a control.

[0214] Having several potent bicyclic quinones in hand, the ability of compounds 7, 9, 12 and 18 to induce apoptosis to DU145 prostate cancer cells was checked, in which USP2 is overexpressed. Following incubation of 6 μΜ concentrations for two hours revealed 3.92% apoptosis for the DMSO control, 50% for β-lapachone, -51% for compound 12, 13% for compound 9, and > 10% for compound 7 (Figure 7). The better and poorer results for compounds 12 and 9, respectively, were in nice correlation with expectations based on their determined ability to inhibit the anti-apoptotic USP2-enzyme' s activity and their ability to affect ROS formation. This does not hold for compound 7, which has excellent inhibitory activity against USP2 and similar redox behavior to that of β-lapachone, but the lowest cytotoxicity within this series.

EXAMPLE 4

CYTOTOXICITY TESTS- ALAMAR BLUE ASSAY General procedure:

[0215] In exemplary procedures, the cytotoxicity assay was performed using alamar blue assay. In a 96 well plates, U87 (glioblastoma), A549 (NSCLC), MCF-7 (breast adenocarcinoma), and Jurkat (acute T cell leukemia) cells lines (harvested under standard buffer conditions) were treated with various concentrations of inhibitor (stock was made using DMSO) and the IC50 values of the inhibitors after 24 and 48 hours were obtained using standard alamar blue protocol.

[0216] As noted hereinabove, compound 7 which exhibits USP2 inhibition, using the quenching pair assay, did not exhibit any significant (9% at 6 μΜ) apoptosis in DU145 cell line. Further cytotoxicity studies using U87 (glioblastoma), A549 (NSCLC), MCF-7 (breast adenocarcinoma), and Jurkat (acute T cell leukemia) cells lines employing alamar blue assay, reveals the IC50 values of 43.8 μΜ, 42.9 μΜ, >100 μΜ and 47.9 μΜ at 24 hours, respectively.

[0217] As noted hereinabove, dimethoxy naphthoquinone, compound 12, which not only retained the USP2 inhibition, but also showed strong apoptosis (51% at 6μΜ) in DU145 cell line. Furthermore, cytotoxicity studies using U87 (glioblastoma), A549 (NSCLC), MCF-7 (breast adenocarcinoma), and Jurkat (acute T cell leukemia) cells lines using alamar blue assay, reveals the IC50 values of 4.2 μΜ, 3.7 μΜ, 27 μΜ and 2 μΜ at 24 hours, respectively.

[0218] The results are summarized in Tables 1 and 2 below [showing the IC50 (μΜ) at 24 hr and at 48 hr, respectively for U87 (glioblastoma), A549 (NSCLC), MCF-7 (breast adenocarcinoma), and Jurkat (acute T cell leukemia)].

Table 1;

EXAMPLE 5

MECHANISM EXAMINATION

[0219] The examinations of DU145 prostate cancer cells, in which USP2 is overexpressed, were tested regarding induced cytotoxicity via treatment with five selected quinones. The results showed that only compound 12 was more potent than β-lapachone (compound 18). This result and the low potency of compound 9 are consistent with their independently acquired information regarding USP2 inhibition, redox potentials, and ROS generation. On the other hand, the same kind of rather naive analysis leads to the expectation that compounds 25 and 7 should also be very cytotoxic, which is clearly not the case. There is no doubt that ROS generation affects the enzymatic activity of USP2, but in more realistic systems there may be more targets for those ROS and their identity might change as a function of the closeness of the particular ROS -generating molecule (naphthoquinones in the present case) to them.

[0220] Without being bound by any particular theory, a reasonable explanation for the larger catalytic activity of o/ /zo-quinone relative to /?ara-quinone for reducing oxygen might be attributed to the stability of the one-electron reduction product obtained in neutral solution, a semiquinone radical. The ortho- but not para- semiquinone radical intermediate may be stabilized by hydrogen bonding of the vicinal oxygen atoms and a proton, via a five-membered ring (Figure 8).

[0221] Without being bound by any particular mechanism, the acidity of this trapped proton should be taken in account when analyzing the reaction with oxygen, by two means: a) it may induce an electron-coupled proton transfer to produce H0 ₂ ' rather than ionized O2 ^" ; and b) it may facilitate the subsequent reduction to hydrogen peroxide (Figure 8).

[0222] On the other hand, reduction of the /?ara-quinone in neutral water solution will produce the non-stabilized semiquinone radical intermediate, which can reduce oxygen only via electron transfer. The produced superoxide anion radical will be relatively stable regarding the second reduction to H2O2, until it reacts with a proton from the solution to produce protonated superoxide radical. That is, the o/ /zo-semiquinone radical intermediate may induce a general acid catalytic effect for the reduction of O2, while catalysis by the para- semiquinone radical intermediate proceeds only via specific acid catalysis.

[0223] An interesting result is the correlation between the redox potentials of the ortho- naphthoquinones, their electro-catalytic activity, and the ability to serve as inhibitors of USP2. The results presented in Table 3 show that the potent inhibitors are very active catalysts for oxygen reduction and that the window of opportunity in terms of the quinone/semiquinone redox potentials is very narrow.

Table 3

1 0 — — —

6 0 0 — —

Para-naphthoquinones

19 11 — — —

24 0 at 0.5 μΜ — — —

[0224] To summarize, systematically investigation of the effect of substituents on the 1,2 naphthoquinone scaffold for beneficial USP2 inhibition is disclosed. Specifically, the quinone/semiquinone redox potentials, and the electrocatalytic reduction of molecular oxygen show a meaningful structure/activity relationships. The comparison of 1,2- and 1,4- naphthoquinone derivatives with identical quinone/semiquinone redox potentials revealed that the former compounds were invariably more potent enzyme inhibitors and better electrocatalysts as well. The latter feature was attributed to a hydrogen -bonding network present in the o/ /zo- semiquinone radicals, which provides the opportunity of general acid catalysis for the reduction of oxygen. Formation of reduced oxygen (the precursor of all ROS) is most significant for compounds within a very narrow range of redox potentials. Optimization of all deduced variables allows to the identification of a new lead compound with beneficial USP2 inhibition and redox properties: the 4 -methoxy- substituted 1,2-naphthoquinone (compound 12). The obtained lead compound 12 possesses simplified structure compared to β-lapachone yet exhibited potent inhibition of USP2 activity. Notably, the effect of substituent's on the quinone ring is more influential on the inhibition of USP2 compared to the substitutions on the aromatic ring. In addition, it was also demonstrated that the mode of inhibition of compound 12 is through the oxidation of catalytic Cys to its sulfinic acid state (Figure 6) and further showed that it induces apoptosis in DU145 cells. Altogether, an efficient strategy that may be applied in other systems that are affected by the generation of ROS.

[0225] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. [0226] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.