WO2018044986A1 - Triazinoindole-based chemical inhibitors of eukaryotic ribosome biogenesis - Google Patents

Abstract

Triazinoindole-based chemical inhibitors of eukaryotic ribosome genesis are disclosed. The compounds have the following structure: The compounds disclosed are useful in treatment of fungal disease and similar diseases associated with the dysregulation of the midasin signaling pathway.

Description

TRIAZINOINDOLE -BASED CHEMICAL INHIBITORS OF

EUKARYOTIC RIBOSOME BIOGENESIS

Cross-reference to Related Applications

[001] This application claims priority from US provisional applications 62/381,355; 62/382,597; and 62/410,605, filed August 30, 2016; September 1, 2016; and October 20, 2016 respectively. All are incorporated herein by reference in their entirety.

Government Rights Statement

[002] This invention was made with government support under NIGMS GM098579 awarded by National Institutes of Health. The government has certain rights in the invention.

Field of the Invention

[003] This invention relates to triazinoindole-based chemical inhibitors of eukaryotic ribosome genesis. The compounds disclosed are useful in treatment of fungal diseases.

BACKGROUND

[004] Every minute a growing cell can generate -2000 ribosomes, ribonucleoprotein- based nanomachines that translate genomes to synthesize proteins (Warner, 1999). The assembly of ribosomes, which contain -5500 nucleotides of RNA and -80 proteins that are together organized into the 60S and 40S subunits, starts in the nucleolus with the transcription of ribosomal DNA. Subsequent steps lead to the processing of RNA to generate 25S, 5.8S, and 5S rRNAs (ribosomal RNAs) for the 60S subunit and the 18S rRNA for the 40S subunit. These steps are coupled to the ordered association and dissociation of several different ribosomal and non-ribosomal proteins to form pre-ribosome subunits that accumulate in the nucleus. Properly assembled pre-ribosomes are then exported to the cytoplasm, where the final processing steps lead to functional ribosomes (Tschochner and Hurt, 2003). Currently, it is unclear how such elaborate spatial and temporal coordination of the several steps required for ribosome assembly is achieved by -200 non-ribosomal proteins (Thomson et al., 2013). This is in large part due to the lack of tools and approaches to analyze this essential and dynamic process. [005] The assembly of the 60S subunit involves multiple intermediates, collectively referred to as pre-60S particles (Nissan et al., 2002). The rearrangements of these particles have been characterized using imaging and immunoprecipitation-based approaches, and the key intermediates are linked to specific proteins that associate with distinct particles, such as the Nsal -associated complex (named the Nsal -particles) that assemble in the nucleolus and the Rixl -associated complex (named the Rixl -particles) that are processed in the nucleoplasm (Kressler et al., 2012). The fidelity of this dynamic assembly process depends on precise spatial and temporal regulation at each stage. For example, the Nug2 GTPase-dependent checkpoint blocks export of pre-60S particles to the cytoplasm until it is properly assembled (Matsuo et al., 2014). The sequential processing of these ribosome assembly intermediates requires numerous energy-consuming enzymes, including ATPases in the AAA+ (ATPases associated with diverse cellular activities) family (Thomson et al., 2013).

[006] The AAA+ family is a large and functionally diverse group of enzymes, that can couple ATP-hydrolysis with mechanical work (e.g. protein unfolding and directional transport). At least three AAA+ ATPases, Drgl, Rix7, and Midasin (Real in S. cerevisiae and Mdnl in S. pombe), are involved in the assembly of the 60S subunit (Kressler et al. Biochim. Biophys. Acta 1823, 92-100. (2012)). Drgl and Rix7 are closely related to the well-studied Cdc48 (p97/VCP in mammals) and likely function as homohexamers with six equivalent ATPase sites. By contrast, Midasin has all six ATPase domains within a single long polypeptide, an organization that is similar to that of the microtubule motor cytoplasmic dynein. Midasin' s ATPase domains form a ring-like domain from which extends an elongated 'tail' with a Metal Ion-Dependent Adhesion Site (MIDAS) domain at its end. Currently, the ATPase activity of Midasin, the largest protein in yeast, has not been characterized and it is unclear if all of its ATPase domains are essential for ribosome biogenesis.

[007] Small molecule inhibitors of midasin would be useful in treatment of fungal disease and similar diseases associated with the dysregulation of the midasin signaling pathway.

BRIEF SUMMARY OF THE INVENTION

[008] In one aspect, the invention relates to compounds of general formula I:

I wherein

R¹ is chosen from -(C₃-Ci₂)hydrocarbyl and -(CH2)heteroaryl, wherein any one of said -(C₃-Ci2)hydrocarbyl or -(CH₂)heteroaryl may be optionally substituted with one or two substituents chosen independently from OH, halogen, nitro, (Ci- C₃)alkylamino, (Ci-C₃)dialkylamino, (Ci-C₃)acylamino, (Ci-C₃)alkylsulfonyl, (Ci- C₃)alkylthio, (Ci-C₃)alkyl, (Ci-C₃)haloalkyl, (Ci-C₃)haloalkoxy, (Ci- C₃)haloalkylthio, -CC(=0)0(Ci-C₃)alkyl, and (Ci-C₃)alkoxy;

R² is halogen, trifluoromethyl, or hydrogen;

R³ is halogen or trifluoromethyl, or when R¹ is other than benzyl, R³ may additionally be hydrogen, wherein at least one of R² and R³ is halogen or trifluoromethyl; and

R⁴ is chosen from hydrogen and -CH₂CH₂C≡CH.

[009] In another aspect, the invention relates to methods of inhibiting midasin comprising contacting midasin with a compound of general formula I

I wherein

R¹ is chosen from -(C₃-Ci₂)hydrocarbyl and -(CH2)heteroaryl, wherein any one of said -(C₃-Ci2)hydrocarbyl or -(CH₂)heteroaryl may be optionally substituted with one or two substituents chosen independently from OH, halogen, nitro, (Ci- C₃)alkylamino, (Ci-C₃)dialkylamino, (Ci-C₃)acylamino, (Ci-C₃)alkylsulfonyl, (Ci- C₃)alkylthio, (Ci-C₃)alkyl, (Ci-C₃)haloalkyl, (Ci-C₃)haloalkoxy, (Ci- C₃)haloalkylthio,

and (Ci-C₃)alkoxy, and wherein said -(C₃- Ci₂)hydrocarbyl is other than benzyl;

R² and R³ are chosen independently from a halogen, trifluoromethyl, and hydrogen; and

R⁴ is chosen from hydrogen and -CH₂CH₂C≡CH.

[010] In another aspect, the invention relates to methods of treating a disease or disorder in a subject where the disease or disorder involves the dysregulation of the midasin signaling pathway, said method comprising administering to the subject a therapeutically effective amount of at least one compound of general formula I:

I wherein

R¹ is chosen from -(C₃-Ci₂)hydrocarbyl and -(CH₂)heteroaryl, wherein any said -(C₃-Ci₂)hydrocarbyl or -(CH₂)heteroaryl may be optionally substituted with one or two substituents chosen independently from OH, halogen, nitro, (Ci- C3)alkylamino, (Ci-C3)dialkylamino, (Ci-C3)acylamino, (Ci-C3)alkylsulfonyl, (Ci- C₃)alkylthio, (Ci-C₃)alkyl, (Ci-C₃)haloalkyl, (Ci-C₃)haloalkoxy, (Ci- C3)haloalkylthio, -CC(=0)0(Ci-C3)alkyl, and (Ci-C3)alkoxy, and wherein said -(C3 Ci2)hydrocarbyl is other than benzyl;

R² and R³ are chosen independently from a halogen, trifluoromethyl, and hydrogen; and

R⁴ is chosen from hydrogen and -CH2CH₂C≡CH.

[Oi l] In another aspect, the invention relates to pharmaceutical compositions comprising at least one compound of general formula I:

I wherein

R¹ is chosen from -(C3-Ci₂)hydrocarbyl and -(CH₂)heteroaryl, wherein any one of said -(C3-Ci₂)hydrocarbyl or -(CH₂)heteroaryl may be optionally substituted with one or two substituents chosen independently from OH, halogen, nitro, (Ci- C3)alkylamino, (Ci-C3)dialkylamino, (Ci-C3)acylamino, (Ci-C3)alkylsulfonyl, (Ci- C₃)alkylthio, (Ci-C₃)alkyl, (Ci-C₃)haloalkyl, (Ci-C₃)haloalkoxy, (Ci- C3)haloalkylthio, -CC(=0)0(Ci-C3)alkyl, and (Ci-C3)alkoxy, and wherein said -(C3- Ci₂)hydrocarbyl is other than benzyl;

R² and R³ are chosen independently from a halogen, trifluoromethyl, and hydrogen; and R⁴ is chosen from hydrogen and -CH2CH₂C=CH; and armaceutically acceptable excipient.

Detailed Description of the Invention

[012] In one aspect, the invention relates to compounds having general formula

I.

[013] In some embodiments of formula I, R¹ may be chosen from -(C3-Ci₂)hydrocarbyl and -(CH₂)heteroaryl, wherein any one of said -(C3-Ci₂)hydrocarbyl or -(CH₂)heteroaryl may be optionally substituted with one or two substituents chosen independently from OH, halogen, nitro, (Ci-C3)alkylamino, (Ci-C3)dialkylamino, (Ci-C3)acylamino, (Ci-C3)alkylsulfonyl, (Ci- C₃)alkylthio, (Ci-C₃)alkyl, (Ci-C₃)haloalkyl, (Ci-C₃)haloalkoxy, (Ci- C₃)haloalkylthio,

, -CC(=0)0(Ci-C3)alkyl, and (Ci-C3)alkoxy. In some embodiments, R² may be halogen or hydrogen. In some embodiments, R³ may be halogen, or when R¹ is other than benzyl, R³ may additionally be hydrogen, wherein at least one of R² and R³ is halogen. In some embodiments, R⁴ may be chosen from hydrogen and -CH₂CH₂C≡CH.

[014] In some embodiments of formula I, R³ may be halogen. In some embodiments, one or both of R² and R³ may be independently Br or I. In some embodiments, R³ may be Br. In some embodiments of formula I, R³ may be trifluorom ethyl.

[015] In some embodiments of formula I, R⁴ may be hydrogen, R¹ may be— CH₂-R⁶ and R⁶ may be chosen from (C2-C₆)alkenyl, (C3-C₆)cycloalkyl, phenyl, substituted phenyl, pyridinyl substituted pyridinyl, pyrazolyl and substituted pyrazolyl. [016] In some embodiments of formula I, R¹ may be chosen from -CH2(C=CH2)alkyl, - CH₂(CH=CH₂), -CH₂(C≡CH),

-((CH₂)i_-6)CH₃, -CH₂(CH)(CH₂)₂ , -CH₂(fluorophenyl), -CH₂(naphthyl), -CH₂(pyridinyl), and - CH₂(pyrazolyl). The -CH₂(naphthyl), -CH₂(pyridinyl), and -CH₂(pyrazolyl) may be optionally substituted. Preferred substituents on pyridine and pyrazole are (Ci-C₃)alkyl.

[017] In some embodiments of formula I, R¹ =CH₂), -(CH₂)₂CH₃

and e

Br; and R⁴ may be H or

[018] In some embodiments, for example, R¹ may be

R² may be H, R³ may be Br, and R⁴ may be H.

[019] In some embodiments of formula I, R¹

, R² may be H, R³ may be Br, R⁴ may be H, and R⁵ may be chosen from -H, -halogen, -N0₂, and -OCH₃.

[020] In some embodiments of formula I, R¹ may be

, R² may be H, R³ may be Br, and R⁴ may be H.

[021] For convenience and clarity, certain terms employed in the specification, examples, and claims are described herein.

[022] Unless otherwise specified, alkyl (or alkylene) is intended to include linear or branched saturated hydrocarbon structures and combinations thereof. Alkyl refers to alkyl groups of from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, s- butyl, t-butyl and the like.

[023] Cycloalkyl is a subset of hydrocarbon and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups include c-propyl, c-butyl, c-pentyl, norbornyl and the like.

[024] Ci to C20 hydrocarbon includes alkyl, cycloalkyl, polycycloalkyl, alkenyl, alkynyl, aryl and combinations thereof. Examples include benzyl, phenethyl, cyclohexylmethyl, adamantyl, camphoryl and naphthyl ethyl. Hydrocarbon or hydrocarbyl refer to any substituent comprised of hydrogen and carbon as the only elemental constituents. Aliphatic hydrocarbons or hydrocarbyls are hydrocarbons or hydrocarbyls that are not aromatic; they may be saturated or unsaturated, cyclic, linear or branched. Examples of aliphatic hydrocarbons or hydrocarbyls include isopropyl, 2-butenyl, 2-butynyl, cyclopentyl, norbornyl, etc. Aromatic hydrocarbons or hydrocarbyls include benzene (phenyl), naphthalene (naphthyl), anthracene, etc.

[025] Unless otherwise specified, the term "carbocycle" is intended to include ring systems in which the ring atoms are all carbon but of any oxidation state. Thus (C3-C12) carbocycle refers to both non-aromatic and aromatic systems, including such systems as cyclopropane, benzene and cyclohexene. Carbocycle, if not otherwise limited, refers to monocycles, bicycles and polycycles. (Cs-Co) Carbopolycycle refers to such systems as norbornane, decalin, indane and naphthalene.

[026] Heterocycle means an aliphatic or aromatic carbocycle residue in which from one to four carbons is replaced by a heteroatom selected from the group consisting of N, O, and S. The nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. Unless otherwise specified, a heterocycle may be non-aromatic (heteroaliphatic) or aromatic (heteroaryl). Examples of heterocycle s include pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran, benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl, when occurring as a substituent), tetrazole, morpholine, thiazole, pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran and the like. Examples of heterocyclyl residues include piperazinyl, piperidinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl, tetrahydrofuryl, tetrahydropyranyl, thienyl (also historically called thiophenyl), benzothienyl, thiamorpholinyl, oxadiazolyl, triazolyl and tetrahydroquinolinyl.

[027] Hydrocarbyloxy refers to groups of from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms attached to the parent structure through an oxygen. Alkoxy is a subset of hydrocarbyloxy and includes groups of a straight or branched configuration. Examples include methoxy, ethoxy, propoxy, isopropoxy and the like. Lower- alkoxy refers to groups containing one to four carbons. The term "halogen" means fluorine, chlorine, bromine or iodine atoms.

[028] Unless otherwise specified, acyl refers to formyl and to groups of 1, 2, 3, 4, 5, 6, 7 and 8 carbon atoms of a straight, branched, cyclic configuration, saturated, unsaturated and aromatic and combinations thereof, attached to the parent structure through a carbonyl functionality. Examples include acetyl, benzoyl, propionyl, isobutyryl and the like. Lower- acyl refers to groups containing one to four carbons. The double bonded oxygen, when referred to as a substituent itself is called "oxo".

[029] Alkoxy or alkoxyl refers to groups of from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms of a straight or branched configuration attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy and the like.

[030] Oxaalkyl refers to alkyl residues in which one or more carbons (and their associated hydrogens) have been replaced by oxygen. Examples include methoxypropoxy, 3,6,9-trioxadecyl and the like. The term oxaalkyl is intended as it is understood in the art [see Naming and Indexing of Chemical Substances for Chemical Abstracts, published by the

American Chemical Society, 2002 edition, If 196, but without the restriction of 127(a)], i.e. it refers to compounds in which the oxygen is bonded via a single bond to its adjacent atoms (forming ether bonds); it does not refer to doubly bonded oxygen, as would be found in carbonyl groups. Similarly, thiaalkyl and azaalkyl refer to alkyl residues in which one or more carbons has been replaced by sulfur or nitrogen, respectively. Examples of azaalkyl include ethylaminoethyl and aminohexyl.

[031] As used herein, the term "optionally substituted" may be used interchangeably with "unsubsti luted or substituted". The term "substituted" refers to the replacement of one or more hydrogen atoms in a specified group with a specified radical. For example, substituted alkyl, aryl, cycloalkyl, heterocyclyl etc. refer to alkyl, aryl, cycloalkyl, or heterocyclyl wherein one or more H atoms in each residue are replaced with halogen, haloalkyl, alkyl, acyl, alkoxyalkyl, hydroxyloweralkyl, carbonyl, phenyl, heteroaryl, benzenesulfonyl, hydroxy, loweralkoxy, haloalkoxy, oxaalkyl, carboxy, alkoxycarbonyl [-C(=0)0-alkyl],

alkoxycarbonylamino [ HNC(=0)0-alkyl], carboxamido [-C(=0) H₂], alkylaminocarbonyl [- C(=0) H-alkyl], cyano, acetoxy, nitro, amino, alkylamino, dialkylamino,

(alkyl)(aryl)aminoalkyl, alkylaminoalkyl (including cycloalkylaminoalkyl), dialkylaminoalkyl, dialkylaminoalkoxy, heterocyclylalkoxy, mercapto, alkylthio, sulfoxide, sulfone, sulfonylamino, alkylsulfinyl, alkylsulfonyl, alkylsulfonylamino, arylsulfonyl, arylsulfonylamino,

acylaminoalkyl, acylaminoalkoxy, acylamino, amidino, aryl, benzyl, heterocyclyl,

heterocyclylalkyl, phenoxy, benzyloxy, heteroaryloxy, hydroxyimino, alkoxyimino, oxaalkyl, aminosulfonyl, trityl, amidino, guanidino, ureido, benzyl oxyphenyl, and benzyloxy. "Oxo" is also included among the substituents referred to in "optionally substituted"; it will be appreciated by persons of skill in the art that, because oxo is a divalent radical, there are circumstances in which it will not be appropriate as a substituent (e.g. on phenyl). In one embodiment, 1, 2 or 3 hydrogen atoms are replaced with a specified radical. In the case of alkyl and cycloalkyl, more than three hydrogen atoms can be replaced by fluorine; indeed, all available hydrogen atoms could be replaced by fluorine. Such compounds (e.g.perfluoroalkyl) fall within the class of "fluorohydrocarbons". To be clear, a generic term may encompass more than one substituent, that is, for example, "haloalkyl" or "halophenyl" refers to an alkyl or phenyl in which at least one, but perhaps more than one, hydrogen is replaced by halogen. In preferred embodiments, substituents are halogen, haloalkyl, alkyl, acyl, hydroxyalkyl, hydroxy, alkoxy, haloalkoxy, oxaalkyl, carboxy, cyano, acetoxy, nitro, amino, alkylamino, dialkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylsulfonylamino arylsulfonyl, arylsulfonylamino and benzyloxy. [032] Substituents Rⁿare generally defined when introduced and retain that definition throughout the specification and in all independent claims.

[033] As used herein, and as would be understood by the person of skill in the art, the recitation of "a compound" - unless expressly further limited - is intended to include salts of that compound. Thus, for example, the recitation "a compound of formula I" as depicted above, in which R¹ is -(C₃-Ci₂)hydrocarbyl or -(CH2)heteroaryl substituted with (Ci-C₃)alkylamino, would also include salts in which the substituent is -N(alkyl)H₂ ⁺ X^", wherein X is any counterion. In a particular embodiment, the term "compound of formula I" refers to the compound or a pharmaceutically acceptable salt thereof.

[034] The term "pharmaceutically acceptable salt" refers to salts whose counter ion derives from pharmaceutically acceptable non-toxic acids and bases. Suitable pharmaceutically acceptable acids for salts of the compounds of the present invention include, for example, acetic, adipic, alginic, ascorbic, aspartic, benzenesulfonic (besylate), benzoic, boric, butyric, camphoric, camphorsulfonic, carbonic, citric, ethanedisulfonic, ethanesulfonic, ethylenediaminetetraacetic, formic, fumaric, glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic, naphthylenesulfonic, nitric, oleic, pamoic, pantothenic, phosphoric, pivalic, polygalacturonic, salicylic, stearic, succinic, sulfuric, tannic, tartaric acid, teoclatic, p- toluenesulfonic, and the like. Suitable pharmaceutically acceptable base addition salts for the compounds of the present invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, arginine, Ν,Ν'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium cations and carboxylate, sulfonate and phosphonate anions attached to alkyl having from 1 to 20 carbon atoms.

[035] It will be recognized that the compounds of this invention can exist in

radiolabeled form, i.e., the compounds may contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Alternatively, a plurality of molecules of a single structure may include at least one atom that occurs in an isotopic ratio that is different from the isotopic ratio found in nature. Radioisotopes of hydrogen, carbon, phosphorous, fluorine, chlorine and iodine include ²H, ³H, ^UC, ¹³C, ¹⁴C, ¹⁵N, ³⁵S, ¹⁸F, ³⁶C1, ¹²⁵I, ¹²⁴I and ¹³¹I respectively. Compounds that contain those radioisotopes and/or other radioisotopes of other atoms are within the scope of this invention. Tritiated, i.e. ³H, and carbon-14, i.e., ¹⁴C, radioisotopes are particularly preferred for their ease in preparation and detectability. Compounds that contain isotopes ^UC, ¹³N, ¹⁵0, ¹²⁴I and ¹⁸F are well suited for positron emission tomography. Radiolabeled compounds of formula I of this invention and prodrugs thereof can generally be prepared by methods well known to those skilled in the art. Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed in the Examples and Schemes by substituting a readily available radiolabeled reagent for a non-radiolabeled reagent.

[036] Persons of skill will readily appreciate that compounds described herein, when appropriately labeled as described above, can be employed in a method of identifying (i.e.

labeling) specific ATPase enzymes, particularly midasin, in the presence of other enzymes, including other ATPase enzymes, for which their affinity is lower. Usually two orders of magnitude difference in affinity will be sufficient to distinguish between enzymes. Using methods well known to persons of skill in the art, specific AAA+ enzymes can be localized in tissues, cells and organelles. A further aspect of the invention described herein is thus a method of identifying and/or localizing specific AAA+ enzymes.

[037] While it may be possible for the compounds of formula I to be administered as the raw chemical, it is preferable to present them as a pharmaceutical composition. According to a further aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt or solvate thereof, together with one or more pharmaceutically carriers thereof and optionally one or more other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The compositions may be formulated for oral, topical or parenteral administration. For example, they may be given intravenously, intraarterially, subcutaneously, and directly into the CNS - either intrathecally or intracerebroventricularly. [038] Formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration. The compounds are preferably administered orally or by injection (intravenous or subcutaneous). The precise amount of compound administered to a patient will be the responsibility of the attendant physician. However, the dose employed will depend on a number of factors, including the age and sex of the patient, the precise disorder being treated, and its severity. Also, the route of administration may vary depending on the condition and its severity. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

[039] Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

[040] It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[041] As used herein, "treatment" or "treating," or "palliating" or "ameliorating" are used interchangeably herein. These terms refers to an approach for obtaining a therapeutic benefit in the form of eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological systems associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. The compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[042] Terminology related to "protecting", "deprotecting" and "protected"

functionalities occurs throughout this application. Such terminology is well understood by persons of skill in the art and is used in the context of processes that involve sequential treatment with a series of reagents. In that context, a protecting group refers to a group which is used to mask a functionality during a process step in which it would otherwise react, but in which reaction is undesirable. The protecting group prevents reaction at that step, but may be subsequently removed to expose the original functionality. The removal or "deprotection" occurs after the completion of the reaction or reactions in which the functionality would interfere. Thus, when a sequence of reagents is specified, as it is in the processes described herein, the person of ordinary skill can readily envision those groups that would be suitable as "protecting groups". Suitable groups for that purpose are discussed in standard textbooks in the field of chemistry, such as Protective Groups in Organic Synthesis by T.W. Greene [John Wiley & Sons, New York, 1991], which is incorporated herein by reference.

[043] A comprehensive list of abbreviations utilized by organic chemists appears in the first issue of each volume of the Journal of Organic Chemistry. The list, which is typically presented in a table entitled "Standard List of Abbreviations", is incorporated herein by reference.

[044] In general, the compounds of the present invention may be prepared by the methods illustrated in the general reaction scheme as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants that are in themselves known, but are not mentioned here. The starting materials are either commercially available, synthesized as described in the examples or may be obtained by the methods well known to persons of skill in the art.

[045] Scheme 1. General synthesis of RBin-1 analogs.

[046] General synthesis scheme: The synthesis method has been adapted from J. Comb. Chem. 2002, 4, 419-428. Briefly, a solution of thiosemicarbazide 2 (3.38 mmol, 1 equivalent) in boiling water was added with stirring to a solution of isatin or substituted isatin 1 (3.38 mmol, 1 equivalent) in boiling ethanol. The resulting mixture was refluxed for 2h to form precipitates of thiosemicarbazone 3, which was isolated by filtration and washed with ethanol.

Thiosemicarbazone 3, was then transferred to a solution of sodium hydroxide (3.38 mmol, 1 equivalent) in water and refluxed overnight. The reaction mixture was then cooled down to room temperature and the pH of the solution was adjusted to 3 with dilute aqueous HCl (1 :3, v/v) to get precipitates of 5. Triazinoindole 5 was then recrystallized from ethanol or washed with hot ethanol to get pure 5 and dried it in air.

[047] Thiosubstitution of 5 was achieved by the addition of the alkyl bromide 6 (0.335 mmol, 0.67 equivalents) to a stirring solution of 5 (0.5 mmol, 1 equivalent) in 0.1 N NaOH (aqueous) and then refluxing the resulting mixture for 2h. The precipitated product was filtered, washed several times with water followed by hot ethanol to get pure 7.

[048] Scheme 2. Synthesis of indole N-subsituted RBin-1 analog Example 21.

Analog 21

[049] 0.3 g of isatin 8 (0.2 mmol, leq) and 0.98 g of Cs₂C0₃ (0.03 mmol, 1.5 eg) were taken in a 25 mL two-neck round bottom flask and flushed N₂ for 30 min. 2 ml of anhydrous DMF was added at 0 °C while stirring. The resulting mixture was stirred for another 15 min at 0 °C and warmed up to room temperature and stirred for another hour. Then 4-bromo-l -butyl (0.06 μί, 3 eq) was added and stirred at 50 °C overnight. After completion of the reaction 10 ml of 0.2 M HC1 was added to the reaction mixture and poured it into a separatory funnel. The product was extracted with 10 ml ethyl acetate (x 2) and dried over sodium sulfate. The solvent was evaporated using rotary evaporator and the product 9 was dried overnight under high vacuum. The product 9 was then purified using silica gel column chromatography using 30% hexane/ethyl acetate.

[050] Appropriate amount of Isatin N-homopropargyl 9 (0.15 mmol, leq),

semithiocarbazide (0.165 mmol, 1.1 eq), K₂C0₃ (0.15 mmol, leq) and 4 ml of water (500 μΐ of ethanol was added to help solubilizing isatin) were stirred and refluxed for 15 h. The reaction mixture was then cooled down to room temperature and acidified with 1 :3 (v/v) HC1 (aq) to pH ~ 2. The precipitate was filtered and washed twice with water and once with ethanol. The product 10 was dried under vacuum.

[051] 13 μΐ of 3-Bromo-2-methyl propene (0.123 mmol, 1 eq) was added to a stirring solution of 0.03 g N-homopropargyl triazinoindole 10 in 0.1 M aqueous NaOH at refluxing temperatures. The resulting mixture was refluxed for 3 hours, then cooled down to room temperature, filtered and washed 3 times with water and once with ethanol. Product example 21 was dried under vacuum.

[052] Specific examples of compounds of the present invention made via the schemes above include:

example

Example 23

[053] General Synthesis Information.

[054] Materials and instrumentation. Materials were purchased from Sigma-Aldrich and used without purification, unless otherwise noted. Reactions were run in capped round bottom flasks stirred with Teflon®-coated magnetic stir bars. Moisture- and air-sensitive reactions were performed in flame-dried round bottom flasks, fitted with rubber septa or glass gas adapters, under a positive pressure of nitrogen. Moisture- and air-sensitive liquids or solutions were transferred via nitrogen-flushed syringe. Where necessary, solutions were deoxygenated by bubbling with nitrogen using a gas dispersion tube. Evaporation of solvents was accomplished by rotary evaporation using a Biichi rotary evaporator, equipped with a dry ice-acetone condenser, at 5-75 mm Hg at temperatures between 35°C and 50°C. Experiments were monitored by thin layer chromatography (TLC) or liquid chromatography mass spectrometry (LC-MS). The maintenance of 30 °C to 150 °C reaction temperatures was accomplished by the use of an oil bath. Products obtained as solids or high boiling oils were dried under vacuum (~1 mmHg).

[055] Chromatography. Purification of compounds where achieved by solica gel column chromatography, when necessary. Analytical TLC was performed using Whatman 250 micron aluminum backed UV F254 precoated silica gel flexible plates. Subsequent to elution, ultraviolet illumination at 254 nm allowed for visualization of UV active materials. Staining with basic potassium permanganate solution allowed for further visualization.

[056] Physical Data. Proton nuclear magnetic resonance spectra (¾ MR) were recorded on Bruker DPX 400 MHz nuclear magnetic resonance spectrometer. Chemical shifts for ¾ MR spectra are reported as δ in units of parts per million (ppm) relative to tetramethylsilane (δ 0.0) using the residual solvent signal as an internal standard or

tetramethylsilane itself: dimethylsulfoxide-i/6 (δ 2.50, quintet) and deuterium oxide-i/2 (δ 4.80, singlet). Multiplicities are given as: s (singlet), d (doublet), t (triplet), or m (multiplet). Coupling constants are reported as a J value in Hertz (Hz). The number of protons (n) for a given resonance is indicated by nH.

[057] Liquid chromatography mass spectral analyses were obtained using a Waters MicroMassZQ mass spectrometer, with an electron spray ionization (ESI) probe, connected to a Waters 2795 HT Separation Module Alliance HT HPLC system running MassLynx (V4.0). The system used a Waters 996 Photodiode Array Detector set to 254 nm for peak detection, and a Symmetry® C18 (3.5 micron) 2.1 x 50 mm column for separation (mobile phase for positive mode: solvent A: water with 0.1% formic acid, solvent B: acetonitrile; mobile phase for negative mode: solvent A: water with 0.1% morpholine, solvent B: acetonitrile). Values are reported in units of mass to charge (m/z).

[058] The models for Midasin function in 60S biogenesis are based in large part on studies of the S. cerevisiae ortholog, Real . The data, together with electron microscopy studies, have led to a model in which ATP -hydrolysis dependent motion of Real 's 'tail' leads to dissociation of Rsa4 from nucleoplasmic pre-60S particles and Ytml from nucleolar pre-60S particles (Kressler et al., op. cit.). However, in order to address Midasin functions in living cells, acute inhibition is necessary to distinguish between direct effects of Midasin inhibition from cumulative effects resulting from defects at earlier stages of ribosome biogenesis. This is particularly important as conventional genetic analyses, using temperature sensitive strains or overexpression of dominant-negative mutants, suppress protein function over several hours, while many steps of ribosome biogenesis are completed within minutes.

[059] Cell-permeable chemical inhibitors can be powerful tools for examining dynamic cellular processes, such as ribosome biogenesis, as the functions of target proteins can be blocked within minutes. A chemical inhibitor that directly targets eukaryotic ribosome assembly factors is diazaborine, an antibacterial compound active at -0.4 mM in S. cerevisiae, a concentration at which selective target inhibition can be difficult to achieve. Moreover, because diazaborine blocks cytoplasmic steps (i.e. pre-60S maturation) of ribosome biogenesis, chemical probes for the several distinct assembly steps that occur in the nucleolus and nucleus may be lacking. Another chemical inhibitor of ribosome assembly factors is lamotrigine, a compound that blocks this process in bacteria.

[060] In the present disclosure, RBins (for ribosome biogenesis inhibitors) are identified as potent, reversible and specific inhibitors of Midasin. Systematic genetic analyses of RBin- sensitivity and RBin-resi stance in fission yeast, along with biochemical characterization of Mdnl 's ATPase activity, indicate that RBins directly and specifically inhibit Mdnl function in vitro and in cells. Live cell imaging, biochemical approaches, and the use of RBins are combined to inhibit or activate Midasin on the timescale of minutes to analyze ribosome assembly dynamics in cells. The present disclosure uncovers a previously uncharacterized function of Midasin in assembling nucleolar Nsal -particles.

[061] To identify cell-permeable chemical probes of essential cellular processes, fission yeast as a model system has been developed that allows for efficient combining of genetic and chemical approaches. In particular, fission yeast strains (named 'MDR-sup' strains) lacking critical factors for multi-drug resistance have been generated and they have been used for chemical screens that mimic synthetic lethal genetic screens. Compounds that reveal enhanced toxicity to strains with a particular mutation, compared to wild-type cells, are likely to be more selective for a single protein target. A compound, example 1, hereinafter called RBin-1, was identified.

[062] Potency was optimized by generating a focused collection of analogs of RBin-1. Twenty-three compounds were synthesized by condensing isatin (or derivatives) with hydrazinecarbothioamide, and further elaborated using halogenated aliphatic and aromatic moieties. This procedure yielded examples 2-23 in >95% purity and 7-65% overall yield. Dose- dependent analyses were used to determine the potency of these analogs in growth assays. The activity of Example 15 against the strain expressing the resistance-conferring mutation {mdnl- F1093L) was substantially reduced, while activity against the more sensitive strain {mdnl- L1113F) was increased relative to the wild-type strain. Together, these tests identify Example 15 as a potent analog of RBin-1 that is likely to have a similar mechanism of action. [063] The fact that compound sensitivity varies significantly by mutations in mdnl indicates that compounds of the invention could directly target Mdnl . As in other AAA+ proteins, ATP -binding in Mdnl is at the interface of two AAA domains and involves Walker A and B motifs from one AAA domain while the arginine finger motif extends from the following AAA domain (e.g. ATP binding at the AAA1 site depends on Walker Al, Walker Bl, and the arginine finger R2 motifs). Sequence alignment of Mdnl homologues across different species, from yeast to human, showed that key motifs required for ATPase activity, including Walker A, Walker B, and Arginine Finger, are highly conserved in AAA2-AAA5 sites. To address which ATP -binding pockets are essential for Mdnl 's functions, an "mdnl-ts complementation assay" was established, in which different Mdnl mutants are overexpressed in an mdnl temperature sensitive (ts) mutant and the growth at the restrictive temperature is measured. The results suggested that the ATPase activity of AAA2, AAA3, AAA4, and AAA5, and ATP -binding ability of AAA6 are likely essential for Mdnl 's cellular functions.

[064] Inhibition of Mdnl 's steady state ATPase activity by RBin-1 and analogs was next examined. Examples 1 and 15 inhibited the ATPase activity by -40% at 1 μΜ. Under similar conditions compounds that were inactive in cell-based assays did not suppress ATPase activity. Increasing concentrations of Example 15 resulted in greater inhibition (EC50: 0.29 μΜ, Hill coefficient: 1.0); the dose-dependent inhibition saturated at -50%, consistent with inhibition of a subset of the multiple ATPase domains in Mdnl . For comparison, almost complete inhibition of the ATPase activity could be achieved by a non-hydrolyzable ATP analog (2 mM).

[065] Full-length recombinant Mdnl-F1093L, containing a mutation that confers resistance to the RBins in cell-based assays was generated. Mdnl-F1093L is an active enzyme whose ATPase activity (1.8 ATP s^"1) is comparable to that of wild-type Mdnl . Importantly, the F1093L mutation in Mdnl also confers resistance to example 15 in the biochemical assay.

Together, these data establish that the physiological target of the compounds described herein is Mdnl, as the same single mutation can suppress inhibition by RBins in both cell-based and in vitro biochemical activity assays.

[066] To examine the kinetics of ribosome assembly inhibition by RBin-1 in living cells, the distribution of pre-60S particles was tracked by imaging Rpl2501-GFP. Upon RBin-1 treatment the Rpl2501-GFP signal accumulated in the nucleolus within 30 min, and reached a maximum level in 90-120 min. Relief from RBin-1 treatment also revealed stepwise reductions in the nucleolar signal. The Rpl2501-GFP signal reduced substantially (-47%) within 5 min of washout, then another -37% between 20-30 min, and -15% between 40-60 min. Also examined were time-dependent defects in the processing of rRNA intermediates in RBin-1 -treated cells. The pre-rRNAs, such as 35S, 27S and 7S pre-rRNA, accumulated only after 60min of RBin-1 treatment, reaching a maximum level in 90-120 min after treatment. Together, these results indicate that RBins inhibit or activate Midasin function on the minutes timescale in fission yeast cells. The step-wise changes in Rpl2501-GFP levels in the nucleolus, observed upon RBin treatment or washout, also suggest that Midasin inhibition can lead to the accumulation of distinct intermediates of ribosome assembly.

[067] Disclosed herein are new chemical inhibitors for Midasin, an essential AAA+ protein required for ribosome biogenesis. These compounds are the first known potent and selective inhibitors of eukaryotic ribosome biogenesis. The availability of matched inhibitor- sensitive and inhibitor-resistant cells allows systematic analysis of dose-dependent target- specific effects of the chemical probe, addressing a major potential limitation of the use of chemical inhibitors to examine cellular mechanisms. Further, the fast timescale of Mdnl inactivation and activation by RBins is well suited to dissect its role in coordinating the spatial and temporal dynamics of ribosome biogenesis.

[068] Eukaryotic ribosome biogenesis involves complex multi-step RNA processing, the recruitment of ribosomal proteins and dynamic associations of multiple non-ribosomal proteins. Briefly, two ribosomal RNA precursors, the 35S and the pre-5S, are transcribed in the nucleolus. The 35S RNA is processed to generate 27S RNA, which is subsequently cleaved to generate 25.5S and 7S RNAs, which contain sequences corresponding to the 25S and 5.8S in the 60S subunit of the mature ribosome. Findings from studies in budding yeast suggest that Mdnl/Real is likely involved in two steps of ribosome biogenesis. The data indicate that many aspects of Mdnl/Real -dependent ribosome biogenesis are likely to be conserved across fungi.

[069] Importantly, the timescale of Midasin inactivation and activation by RBin-1 (5-15 min) is significantly faster than what can be achieved using genetic approaches (-3-6 h using the temperature-sensitive allele). In particular, the fast temporal control over target function that can be achieved with these chemical probes is evident from studies of Rpl2501 and Rix7 localization, which changes in live cells within 5 min of inhibitor washout.

[070] Characterization of the Examples.

[071] RBin-1 or example 1 (AP-5): ¹H MR (400 MHz, DMSO-d₆) δ 1.84 (s, 3H), 3.98 (s, 2H), 4.91 (s, IH), 5.1 (s, IH), 7.42 (t, J= 7.38 Hz, IH), 7.56 (d, J= 8.0 Hz, IH), 7.68 (t, J= 7.54 Hz, IH), 8.29 (d, J= 7.64 Hz, IH). ESI-MS (m/z) [M+H]+ Calculated: 257.33, found: 257.57.

[072] Example 2 (ZC 1-009): ¾ NMR (400 MHz, DMSO-d₆) δ 3.96 (d, J= 6.68 Hz, 2H), 5.16 (d, J= 9.84 Hz, IH), 5.39 (d, J= 16.8 Hz, IH), 6.04 (m, IH), 7.43 (t, J= 7.46 Hz, IH), 7.52 (d, J= 8.0 Hz, IH), 7.68 (t, J= 7.58 Hz, IH), 8.30 (d, J= 8.0 Hz, IH). ESI-MS (m/z)

[M+H]+ Calculated: 243.30, found: 243.55.

[073] Example 3 (ZC11): ¾ MR (400 MHz, DMSO-d₆) δ 1.03 (t, J= 7.28 Hz, 3H), 1.77 (m, 2H), 3.24 (t, J= 7.16 Hz, 2H), 7.43 (t, J= 7.44 Hz, IH), 7.56 (d, J= 8.04 Hz, IH), 7.68 (t, J= 7.6 Hz, IH), 8.29 (d, J= 7.68 Hz, IH). ESI-MS (m/z) [M+H]+ Calculated: 245.32, found: 245.57.

[074] Example 4 (AP3-89): ¾ NMR (400 MHz, DMSO-d₆) δ 3.18 (s, IH), 4.13 (s, 2H), 7.44 (t, J= 7.4 Hz, IH), 7.58 (d, J= 8.04 Hz, IH), 7.7 (t, J= 7.52 Hz, IH), 8.32 (d, J= 7.64 Hz, IH). ESI-MS (m/z) [M+H]+ Calculated: 241.28, found: 241.53.

[075] Example 5 (ZCl-51): ¾ NMR (400 MHz, DMSO-d₆) δ 0.37 (m, J= 4.81 Hz, 2H), 0.58 (m, J= 5.9 Hz, 2H), 1.24 (m, IH), 3.23 (d, J= 7.12 Hz, 2H), 7.43 (t, J= 7.43 Hz, IH), 7.56 (d, J= 8.08 Hz, IH), 7.68 (t, J= 7.48 Hz, IH), 8.30 (d, J= 7.7 Hz, IH). ESI-MS (m/z)

[M+H]+ Calculated: 257.33, found: 257.59.

[076] Example 6 (AP-13): ¾ NMR (400 MHz, DMSO-d₆) δ 4.55 (s, 2H), 7.25 (t, J= 7.16 Hz, IH), 7.32 (t, J= 7.22 Hz, 2H), 7.42 (t, J= 7.42 Hz, IH), 7.51 (d, J= 7.24 Hz, 2H), 7.56 (d, J= 8.04 Hz, IH), 7.68 (t, J= 7.5 Hz, IH), 8.29 (d, J= 7.64 Hz, IH). ESI-MS (m/z) [M+H]+ Calculated: 293.36, found: 293.62. [077] Example 7 (AP-12): ¾ NMR (400 MHz, DMSO-d₆) δ 4.56 (s, 2H), 7.42 (t, J= 7.48 Hz, IH), 7.52 (d, J= 4.96 Hz, 2H), 7.56 (d, J= 8.08 Hz, IH), 7.68 (t, J= 7.62 Hz, IH), 8.29 (d, J= 7.72 Hz, IH), 8.50 (d, J= 3.72 Hz, 2H). ESI-MS (m/z) M+H]+ Calculated: 294.35, found: 294.59.

[078] Example 8 (ZC-10): ¾ NMR (400 MHz, DMSO-d₆) δ 4.74 (s, 2H), 7.43 (t, J= 7.54 Hz, IH), 7.49 (t, J= 3.69 Hz, 2H), 7.58 (d, J= 8.08 Hz, IH), 7.6 (m, 2H), 7.88 (d, J= 8.08 Hz, 3H), 8.04 (s, IH), 8.30 (d, J= 7.6 Hz, IH). ESI-MS (m/z) M+H]+ Calculated: 243.42, found: 243.65.

[079] Example 9 (ZC l-5): ¾ NMR (400 MHz, DMSO-de) δ 3.73 (s, 3H), 4.53 (s, 2H), 6.83 (d, J= 7.2 Hz, IH), 7.08 (d, J= 7.12 Hz, 2H), 7.24 (t, J= 7.9 Hz, IH), 7.43 (t, J= 7.48 Hz, IH), 7.58 (d, J= 8.04 Hz, IH), 7.69 (t, J= 7.7 Hz, IH), 8.31 (d, J= 7.6 Hz, IH). ESI-MS (m/z) [M+H]+ Calculated: 323.39, found: 323.67.

[080] Example 10 (ZC-41): ¾ NMR (400 MHz, DMSO-d₆) δ 4.86 (s, 2H), 7.43 (t, J= 7.52 Hz, IH), 7.56 (m, 2H), 7.7 (t, J = 7.72, 2H), 7.90 (d, J= 7.72 Hz, IH), 8.05 (d, J= 8.12 Hz, IH), 8.30 (d, J= 7.68 Hz, IH). ESI-MS (m/z) [M+H]+ Calculated: 338.36, found: 338.61.

[081] Example 11 (ZC-7): ¾ NMR (400 MHz, DMSO-d₆) δ 4.69 (s, 2H), 7.44 (t, J= 7.6 Hz, IH), 7.5 (d, J= 8.0 Hz, IH), 7.69 (t, J= 7.6 Hz, IH), 7.80 (d, J= 8.16 Hz, 2H), 8.17 (d, J= 8.16 Hz, 2H), 8.30 (d, J= 7.68 Hz, IH). ESI-MS (m/z) M+H]+ Calculated: 338.36, found:

338.60.

[082] Example 12 (ZC-30): ¾ NMR (400 MHz, DMSO-d₆) δ 4.59 (s, 2H), 7.16 (t, J= 7.44 Hz, IH), 7.22 (t, J= 9.28 Hz, IH), 7.33 (m, IH), 7.43 (t, J= 7.48 Hz, IH), 7.58 (d, J= 8.08 Hz, IH), 7.64 (t, J= 7.62 Hz, IH), 7.69 (t, J= 7.64 Hz, IH), 8.31 (d, J= 7.72 Hz, IH). ESI-MS (m/z) [M+H]+ Calculated: 311.35, found: 311.57.

[083] Example 13 (AP-42): ¾ NMR (400 MHz, DMSO-d₆) δ 4.63 (s, 2H), 7.04 (t, J= 7.5 Hz, IH), 7.36 (t, J= 7.4 Hz, IH), 7.43 (t, J = 7.5, IH), 7.58 (d, J= 8.04 Hz, IH), 7.69 (t, J= 6.94 Hz, 2H), 7.90 (d, J= 7.68 Hz, IH), 8.31 (d, J = 7.64 Hz, IH). ESI-MS (m/z) [M+H]+ Calculated: 419.26, found: 419.48. [084] Example 14 (AP-43): ¾ NMR (400 MHz, DMSO-d₆) δ 4.51 (s, 2H), 7.33 (d, J = 7.76 Hz, 2H), 7.43 (t, J= 7.46 Hz, IH), 7.57 (d, J= 8.0 Hz, IH), 7.68 (t, J= 8.98 Hz, 3H), 8.30 (d, J= 7.6 Hz, IH). ESI-MS (m/z) [M+H]+ Calculated: 419.26, found: 419.55.

[085] Example 15 (ZC-32): ¾ NMR (400 MHz, DMSO-d₆) δ 1.84 (s, 3H), 3.98 (s, 2H), 4.91 (s, IH), 5.12 (s, IH), 7.58 (d, J= 8.28 Hz, IH), 7.74 (s, IH), 8.24 (d, J= 8.4 Hz, IH). ESI-MS (m/z) [M]+ Calculated: 335.22, found: 335.45 and [M+2H]+ Calculated: 337.22, found: 337.48 (bromine pattern).

[086] Example 16 (ZC-33): ¾ MR (400 MHz, DMSO-d₆) δ 4.56 (s, 2H), 7.26 (t, J = 7.24 Hz, IH), 7.33 (t, J= 7.36 Hz, 2H), 7.51 (d, J= 7.36 Hz, 2H), 7.59 (d, J= 7.24 Hz, IH), 7.75 (s, IH), 8.24 (d, J= 8.32 Hz, IH). ESI-MS (m/z) [M]+ Calculated: 371.26, found: 371.49 and [M+2H]+ Calculated: 373.26, found: 373.48 (bromine pattern).

[087] Example 17 (ZC-52): ¾ NMR (400 MHz, DMSO-d₆) δ 0.37 (d, J = 4.12 Hz, 2H), 0.58 (d, J= 7.2 Hz, 2H), 1.24 (m, IH), 3.23 (d, J= 7.04 Hz, 2H), 7.58 (d, J= 8.12 Hz, IH), 7.73 (s, IH), 8.24 (d, J= 8.0 Hz, IH). ESI-MS (m/z) [M]+ Calculated: 335.22, found: 335.52 and [M+2H]+ Calculated: 337.22, found: 337.55 (bromine pattern).

[088] Example 18 (AP-45): ¾ NMR (400 MHz, DMSO-d₆) δ 4.63 (s, 2H), 7.04 (t, J = 7.52 Hz, IH), 7.36 (t, J= 7.4 Hz, IH), 7.59 (d, J= 8.28 Hz, IH), 7.7 (d, J= 7.56 Hz, IH), 7.76 (s, IH), 7.90 (d, J= 7.84 Hz, IH), 8.25 (d, J= 8.2 Hz, IH). ESI-MS (m/z) [M]+ Calculated: 497.15, found: 497.43 and [M+2H]+ Calculated: 499.15, found: 499.46 (bromine pattern).

[089] Example 19 (AP-46): ¾ NMR (400 MHz, DMSO-d₆) δ 4.5 (s, 2H), 7.33 (d, J = 8.08 Hz, 2H), 7.59 (d, J= 8.28 Hz, IH), 7.67 (d, J= 8.16 Hz, 2H), 7.75 (s, IH), 8.24 (d, J= 8.2, IH). ESI-MS (m/z) [M]+ Calculated: 497.15, found: 497.44 and [M+2H]+ Calculated: 499.15, found: 499.45 (bromine pattern).

[090] Example 20 (AP3-91): ¾ NMR (400 MHz, DMSO-d₆) δ 3.18 (s, IH), 4.13 (s, 2H), 7.60 (d, J= 8.24 Hz, IH), 7.74 (s, IH), 8.26 (d, J= 8.28 Hz, IH). ESI-MS (m/z) [M]+ Calculated: 319.18, found: 319.49 and [M+2H]+ Calculated: 321.18, found: 321.51 (bromine pattern). [091] Example 21 (AP-38): ¾ NMR (400 MHz, DMSO-de) δ 2.78 (m, 3H), 4.0 (s, 2H), 4.55 (t, J= 6.66, 2H), 4.91 (s, 1H), 5.14 (s, 1H), 7.48 (t, J= 7.46 Hz, 1H), 7.75 (t, J= 7.44 Hz, 1H), 7.90 (d, J= 8,24 Hz, 1H), 8.34 (d, J= 7.64 Hz, 1H). ESI-MS (m/z) [M+H]+ Calculated: 309.40, found: 309.65.

[092] Example 22 (ZC-21): ¾ NMR (DMSO) δ 1.85 (s, 3H), 3.99 (s, 2H), 4.91 (s, 1H), 5.12 (s, 1H), 7.42 (d, J= 8.4 Hz, 1H), 7.96 (d, J= 8.4 Hz, 1H), 8.59 (s, 1H). ESI-MS (m/z) [M+H]+ Calculated: 383.22, found: 383.51.

[093] Example 23 (ZCl-25): ¾ NMR (400 MHz, DMSO-de) δ 4.56 (s, 2H), 7.25 (t, J = 7.04 Hz, 1H), 7.33 (t, J= 7.32 Hz, 2H), 7.42 (d, J= 8.4 Hz, 1H), 7.51 (d, J = 7.36 Hz, 2H), 7.96 (d, J= 8.56 Hz, 1H), 8.60 (s, 1H). ESI-MS (m/z) [M+H]+ Calculated: 419.26, found:

419.48.

[094] Antifungal SAR analysis of examples using growth assay of

Schizosaccharomyces pombe. S.pombe strains were grown to exponential phase in 5g of yeast extract (Fisher BioReagents), 30 g of Dextrose Anhydrous Powder (J.T. Baker), 250 mg of L- Histidine (Sigma- Aldrich), 250 mg of L-Leucine (Sigma- Aldrich), 250 mg of adenine

hemisulfate salt (Sigma-Aldrich) and 250 mg of uridine per liter of solution. The samples were diluted to ODeoo-0.01, and 1 μL· DMSO or 1μΙχ>ί a DMSO solution of test compound (lOOOx) were mixed with 1 μΙ_, diluted cell suspensions. The cells were placed in shaker (220 rpm) for 18 hr at 29°C till ODeoo of DMSO control reached ~1. Relative growth was calculated by dividing the measured OD at a specific concentration by the OD for the DMSO control. Half maximum growth inhibition (GI50) was determined by fitting relative growth to a four-parameter sigmoidal dose-response curve in PRISM.

Results are shown in Table 1 below.

Table 1.

Examples R¹ R² R³ R⁴ GIso (μΜ)

23 I H H > 2.5

24 H F H 0.208

25 H CF₃ H 0.163

26 H Br H 0.147

27 H CF₃ H 0.138

\

CH₃

28 H CI H 0.082

\

CH₃

29

H Br H >0.100

\

CH₃ Examples R¹ R² R³ R⁴ GIso (μΜ)

30 H CI H >0.100

[095] Purification of recombinant Schizosaccharomyces pombe Mdnl

[096] All biochemical reagents were obtained from SIGMA-ALDRICH unless specified otherwise. The cDNA encoding fission yeast Mdnl was cloned into pFastBac HTC vector (Invitrogen). We used the Bac-to-Bac system (Invitrogen) to generate recombinant baculovirus. High Five cells (Life Technologies) were grown in Sf-900 II SFM (Life Technologies 10902- 096) with IX Antibiotic- Antimyocotic (Life Technologies) to -2.5 million/mL and then infected (1 :80 dilution of P2 virus). The cells were harvested 60 hr after infection. All of the following steps were done on ice or at 4°C. The cells were lysed by sonication in equal volume of lysis buffer (50mMTris, 400mM NaCl, 20 mM imidazole, 1 mM MgCb, 5 μΜ 2-mercaptoethanol, 200 μΜ ATP, 3 U/mL benzonase, IX Roche complete protease inhibitor without EDTA, 10% glycerol [pH 7.5]). The homogenized lysate was then centrifuged at 55,000 rpm for 1 hr. The supernatant was incubated with Ni-NTA beads (QIAGEN) for 40 min. The beads were extensively washed using Washing buffer (50 mM Tris, 400 mM NaCl, 20 mM imidazole, 1 mM MgCb, 5 μΜ 2-mercaptoethanol, 10% glycerol [pH 7.5]). The protein was then eluted by high imidazole buffer (20 mM Tris [pH 7.5], 120 mM NaCl, 300 mM imidazole, 1 mM MgCb, 5 μΜ 2-mercaptoethanol, 200 μΜ ATP (A2383)). The eluted fraction was filtered and loaded onto a Mono Q column 5/50 GL (GE Healthcare Life Sciences). The protein was eluted around 400 mM NaCl and the fractions were collected and analyzed by SDS-PAGE. The relevant fractions were pooled and then concentrated using Amicon Ultra-4 Centrifugal Filter Units. The concentrated sample was then loaded on Superose 6, 10/300 GL (GE Healthcare Life Sciences) using FPLC SEC buffer (20 mM Tris (pH = 7.5), 150 mM NaCl, 1 mM MgCb, ImM EGTA and 5 μΜ 2-mercaptoethanol). Peak fractions were collected and ATPase assay was carried out directly with fresh protein. Protein concentration was determined using a Bradford assay. [097] Radioactive ATPase Assay

[098] Radioactive γ-Ρ32-ΑΤΡ (PerkinElmer, BLU002Z250UC) was added to 600 μΜ MgATP (pH = 7.0) solutions at volume ratios of 1 : 1000-1 :300, depending on the lifetime of the radioactive reagent. The total volume of each reaction was 12 μΐ_^, including 6 μΐ of protein from size exclusion chromatography fractions (final concentration -0-50 nM for different fractions, peak fractions were used for Rbin-1 and AMPP P inhibition experiments in Figure 3E), 4 μΐ FPLC SEC buffer with 0.6 mM Na₂S0₄ and 2 μΐ MgATP (final concentration = 100 μΜ). The reactions were then incubated at room temperature for 30 or 60 min before quenching with 12 μΐ 0.2 M EDTA. 1 μΐ from each reaction mixture was spotted on to TLC PEI cellulose F plates (Millipore, 105579). The TLC buffer contained 0.15 M formic acid and 0.15 M lithium chloride. The TLC plates were then imaged using the Typhoon Scanner 9400 (GE Healthcare Life Sciences). ImageJ was used to calculate the densitometric ratio of the spots corresponding to radioactive free phosphate and ATP to determine the percent of ATP hydrolyzed.

Claims

CLAIMS What is claimed is:

1. A compound of formula I:

I

wherein

R¹ is chosen from -(C3-Ci2)hydrocarbyl and -(CH2)heteroaryl, wherein any one of said -(C3-Ci2)hydrocarbyl or -(CH2)heteroaryl may be optionally substituted with one or two substituents chosen independently from OH, halogen, nitro, (G- C3)alkylamino, (G-C3)dialkylamino, (Ci-C3)acylamino, (G-C3)alkylsulfonyl, (G- C₃)alkylthio, (Ci-C₃)alkyl, (G-G)haloalkyl, (Ci-C3)haloalkoxy, (G- C₃)haloalkylthio, -CC(=0)0(G-C₃)alkyl, and (Ci-C₃)alkoxy;

R² is halogen, trifluoromethyl or hydrogen;

R³ is halogen or trifluoromethyl, or, when R¹ is other than benzyl, R³ may additionally be hydrogen, with the proviso that at least one of R² and R³ must be halogen or trifluoromethyl; and

R⁴ is chosen from hydrogen and -CH2CH2C≡CH.

2. The compound of claim 1, wherein R³ is halogen or CF3.

3. The compound of claim 1 or 2 wherein R⁴ is hydrogen, R¹ is -CH2-R⁶ R⁶ is chosen from (C2-C₆)alkenyl, (C3-C₆)cycloalkyl, phenyl, substituted phenyl, pyridinyl substituted pyridinyl, pyrazolyl and substituted pyrazolyl.

4. The compound of claim 1 or 2, wherein

R¹ is chosen from -CH₂(C=CH₂)alkyl, -CH₂(CH=CH₂), -CH₂(C≡CH), - ((CH₂)_1-6)CH₃, -CH₂(CH)(CH₂)₂ , -CH₂(fluorophenyl)_, -CH₂(naphthyl), - CH₂(pyridinyl), and -CH₂(pyrazolyl) wherein said -CH₂(naphthyl), - CH₂(pyridinyl), and -CH₂(pyrazolyl) may be optionally substituted.

5. The compound of claim 1 or 2, wherein

R¹ is chosen from -CH₂(C=CH₂)CH₃, -CH₂(CH=CH₂), -(CH₂)₂CH₃,

R² is H; R³ is Br; and

6. The compound of claim 1 or 2, wherein

R² is H; R³ is Br; and R⁴ is H.

7. The compound of claim 1 or 2, wherein

R² is H; R³ is Br; R⁴ is H; and

R⁵ is chosen from -H, -halogen, -NO2, and -OCH3.

8. The compound of claim 1 or 2, wherein

is H;

R³ is Br; and is H.

9. A method of inhibiting midasin comprising contacting midasin with a compound of formula I:

wherein R¹ is chosen from -(C3-Ci2)hydrocarbyl and -(CH2)heteroaryl, wherein any one of said -(C3-Ci2)hydrocarbyl or -(CH2)heteroaryl may be optionally substituted with one or two substituents chosen independently from OH, halogen, nitro, (Ci- C3)alkylamino, (Ci-C3)dialkylamino, (Ci-C3)acylamino, (Ci-C3)alkylsulfonyl, (Ci- C₃)alkylthio, (Ci-C₃)alkyl, (Ci-C₃)haloalkyl, (Ci-C₃)haloalkoxy, (G- C₃)haloalkylthio, -CC(=0)0(Ci-C₃)alkyl, and (Ci-C₃)alkoxy, and wherein said -(C₃- Ci2)hydrocarbyl is other than benzyl;

R² and R³ are chosen independently from a halogen, trifluoromethyl, and hydrogen; and

R⁴ is chosen from hydrogen and -CH2CH2C≡CH.

10. A method according to claim 9 wherein said method of inhibiting is an in vitro method.

11. A method for treating a disease or disorder in a subject where the disease or disorder involves the dysregulation of the midasin signaling pathway, said method comprising administering to the subject a therapeutically effective amount of a compound of formula I:

I wherein

R¹ is chosen from -(C3-Ci2)hydrocarbyl and -(CH2)heteroaryl, wherein any one of said -(C3-Ci2)hydrocarbyl or -(CH2)heteroaryl may be optionally substituted with one or two substituents chosen independently from OH, halogen, nitro, (G- C3)alkylamino, (Ci-C3)dialkylamino, (Ci-C3)acylamino, (G-C3)alkylsulfonyl, (G- C₃)alkylthio, (Ci-C₃)alkyl, (Ci-C₃)haloalkyl, (Ci-C₃)haloalkoxy, (Ci- C₃)haloalkylthio, -CC(=0)0(Ci-C₃)alkyl, and (Ci-C₃)alkoxy, and wherein said -(C₃- Ci2)hydrocarbyl is other than benzyl;

R² and R³ are chosen independently from a halogen, trifluoromethyl, and hydrogen; and

R⁴ is chosen from hydrogen and -CH2CH2C≡CH.

12. A method according to claim 1 1 wherein the disease or disorder is a fungal disease.

13. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of formula I:

I wherein

R¹ is chosen from -(C₃-Ci2)hydrocarbyl and -(Ctyheteroaryl, wherein any one of said -(C3-Ci2)hydrocarbyl or -(CH2)heteroaryl may be optionally substituted with one or two substituents chosen independently from OH, halogen, nitro, (Ci-C₃)alkylamino, (Ci- C₃)dialkylamino, (Ci-C₃)acylamino, (Ci-C₃)alkylsulfonyl, (Ci- C₃)alkylthio, (Ci-C₃)alkyl, (Ci-C₃)haloalkyl, (Ci-C₃)haloalkoxy, (Ci- C₃)haloalkylthio, -CC(=0)0(Ci-C₃)alkyl, and (Ci-C₃)alkoxy, and wherein said -(C₃-Ci2)hydrocarbyl is other than benzyl; R² and R³ are chosen independently from halogen, trifluoromethyl, and hydrogen; and

R⁴ is chosen from hydrogen and -CH₂CH2C≡CH.

14. An oral pharmaceutical composition according to claim 13.