JP2015510886A - Selective histone deacetylase 6 inhibitor - Google Patents

Abstract

Disclosed is a selective histone deacetylase inhibitor (HDACi) having the formula (I). Also disclosed are methods of making and using these inhibitors for the treatment of cancer, particularly melanoma. [Chemical 1] [Selected figure] None

Description

Confirmation of Government Support This invention was made by the National Institutes of Health under grant number CA 134807 with government support. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS This application is hereby incorporated by reference in its entirety, US Provisional Patent Application No. 61 / 607,083 filed March 7, 2012; filed March 30, 2012 US Provisional Patent Application No. 61 / 618,150; US Provisional Patent Application No. 61 / 648,946 filed May 18, 2012; US Provisional Patent Application No. 61 / 648,946 filed May 25, 2012 61 / 651,595; US provisional patent application 61 / 651,896 filed May 25, 2012; US provisional patent application 61 / 674,942 filed July 24, 2012 And claims the benefit of priority according to US Provisional Patent Application No. 61 / 715,379, filed Oct. 18, 2012.

Epigenetic regulation and subsequent gene expression or silencing tightly regulates interactions between enzymes that serve to modify the histone tail around which nuclear DNA wraps. Among the various modifiers of histones, cells have the activity of both histone acetyltransferase (HAT) and histone deacetylase (HDAC) to bind or remove acetyl groups, respectively, from the lysine tails of these histone cylinders. Can be balanced. This particular epigenetic marker shields positive lysine residues from interacting closely with the phosphate-backbone of DNA, resulting in a more “open” chromatin state, while deacetylase removes these acetyl groups. This results in a more “closed” or compressed DNA-histone state.

Currently, there is no selective HDAC6 inhibitor (HDAC6i) approved for oncological purposes. These molecules may be advantageous as therapeutic approaches because they may lead to reduced side effects, an obvious problem associated with relatively low selectivity HDACIs (Zhang et al., “Mice lacquering histone deacetylase 6 have hyperhyperlated). "tubulin but are viable and develop normally", Mol Cell Biol, 2008, 28 (5): 1688-1701). Recent preclinical efforts have been directed to the use of HDAC6i for certain cancers, particularly in combination with known drugs (Santo et al., “Preclinical activity, pharmacodynamic, and pharmacokinetic properties HD6A6). , ACY-1215, in combination with bortezomib in multiple myeloma ”, Blood 2012, 119 (11): 2579-2589). HDACIs may be useful as possible therapeutic agents for melanoma; however, studies to date have focused on the use of pan-HDACI such as suberoylanilide hydroxamic acid (SAHA) (Peltonen et al. , “Melanoma cell lines are susceptible to histone deacetylase inhibitor TSA-provoked cell cycle array and apoptosis”, Pigment Cell Res 2005, 183; human melanoma cel s exposed to histone deacetylase inhibitors ", Apoptosis 2004, (5): 573-582). SAHA exhibits activity against all Zn-dependent HDAC isoenzymes, but has been exclusively approved for the treatment of cutaneous T-cell lymphomas (Wagner et al., “Histone deacetylase (HDAC) inhibitors in recurrent clinical trials). "Clinical Epigenetics 2010, 1 (3-4): 117-136). HDAC6 has previously been reported to form an association with HDAC11 (Gao et al., “Cloning and functioning of HDAC11, a novel member of the human histone family 2”, J : 25748-25755). Recent efforts have begun to reveal the biological significance of HDAC11 as a participant in the activation of immune responses, and targeting one or both of these enzymes has therapeutic value (Villagra et al. . al, "The histone deacetylase HDACl 1 regulates the expression of interleukin 10 and immune tolerance", Nat Immunol 2009,10 (1):. 92-100; Wang et al, "Histone Deacetylase Inhibitor LAQ824 Augments Inflammatory Responses in Macrophages through Transcript ion Regulation of IL-10 ", J Immunol 2011, 186 (7): 3986-3996). Therefore, HDAC6 has emerged as a target in the treatment of melanoma and other cancers. Such an approach is valuable in the context of safer cancer therapeutics because it may lack pan-HDACi cytotoxicity (Parmigani et al., “HDAC6 is a specific decades of peroxyredoxins and is involved in redox regulation ", Proc Nat Acad Sci USA 2008, 705 (28): 9633-9638). There is a need for new and selective HDAC6 inhibitors and methods for their production and use to treat various cancers and enhance various tumor immune responses. The compositions and methods disclosed herein address these and other needs.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

It is an image showing the classification of HDAC. It is an image which shows that HDAC is a target of a histone deacetylase inhibitor (HDACi). FIG. 6 is an image showing that HDAC6 was found to affect IL-10 gene expression within APC. It is an image which shows that the gene or pharmacological disruption of HDAC6 inhibits IL-10. It is an image which shows that the gene disruption of HDAC6 improves APC function. The mechanism shown by CHIP analysis of the IL-1O gene promoter in macrophages involves H3 and H4 acetylation; HDAC6 recruitment; and binding of STAT3 and other transcription factors at several time points after LPS stimulation It is an image which shows. FIG. 6 is an image showing that knockdown of HDAC6 results in reduced recruitment of the transcriptional activator STAT3 to the IL-10 gene promoter. FIG. 5 is an image showing that disruption of STAT3 binding to the gene promoter resulted in reduced recruitment of HDAC6 and reduced IL-10 production. It is an image which shows that destruction of HDAC6 inhibits STAT3 phosphorylation. 1 is a series of images showing increased expression of HDAC6 and IL-10 mRNA in human melanoma. FIG. 6 is a series of images showing HDAC6 expression in mouse and human melanoma cell lines. Figure 2 is a series of images showing HDAC protein expression in melanoma. FIG. 6 is a series of images showing reduced proliferation and cell cycle arrest in melanoma cells lacking HDAC6. A series of images showing that melanoma cells lacking HDAC6 are more immunogenic. 2 is a series of images showing that pharmacological inhibition of HDAC6 in melanoma cells resulted in cell cycle arrest and increased expression of MHC molecules. 3 is an image showing that Tubastatin A inhibits JAK2 / STAT3 phosphorylation in B16 mouse melanoma cells in vivo. 2 is a series of images showing that Tubastatin A enhances antigen-specific CD4 + T cell responses to vaccination in melanoma-supported mice. FIG. 3 is an image showing that Tubastatin A, a selective HDAC6 inhibitor, reduced STAT3 phosphorylation and recruitment to the IL-10 gene promoter in APC. FIG. 2 is an image showing phenotype and functional changes in APC treated with Tubastatin A. FIG. It is an image showing that Tubastatin A-treated APC is a better activator of naive T cells and restores responsiveness of response-invisible T cells. It is a graph which shows the antitumor effect of Tubastatin A in vivo. FIG. 3 is a series of images showing that Tubastatin A does not affect PEM. 1 is a series of images showing the immunological effect of Tubastatin A on macrophages. FIG. 3 is a series of images showing that Tubastatin A inhibits IL-10 transcription by disrupting the JAKISTAT3 pathway in macrophages. FIG. 6 is a graph showing that the inhibitory effect of Tubastatin A on IL-10 production is lost in the absence of HDAC6. It is a flowchart which shows the experimental design of the antigen presentation test in vitro. FIG. 6 is a series of images showing that Tubastatin A-treated macrophages are better activators of naive T cells and restore function of response-invisible T cells. 2 is a series of images showing that treatment with Tubastatin A in vivo enhances the response of antigen-specific T cells to vaccination. It is an image which shows HDAC6 expression in human MCL. FIG. 6 is a series of images showing the destruction of HDAC6 in a human MCL cell line. It is an image which shows destruction of HDAC6 in a mouse | mouth FC-muMCLI cell. It is an image which shows the immunological effect of HDAC6 inhibition in MCL. FIG. 6 shows changes in MHC, costimulatory molecules and / or cytokine production in response to LPS or CpG +/− ST-3-06 or Tubastatin A. FIG. It is a series of images showing the antigen presenting function of FCmuMCLI cells treated with ST-3-06. It is a series of images showing the antigen presenting function of FCmuMCLI cells treated with Tubastatin A. It is an image which shows the antitumor effect of Tubastatin A in vivo. FIG. 6 is a series of images showing that HDAC6 disruption inhibits STAT3 phosphorylation within APC. It is a series of images showing that ST-3-06 reduced STAT3 phosphorylation and recruitment to the IL-10 gene promoter in APC. Western blot showing 5 g of substrate specificity.

In accordance with the purpose of the disclosed materials, compounds, compositions, articles, devices and methods embodied and broadly described herein, the disclosed subject matter is a composition and methods of making and using the composition. About. In another aspect, the disclosed subject matter relates to compounds having activity as selective HDAC6 inhibitors, methods of making and using the compounds, and compositions comprising the compounds. In certain embodiments, the disclosed subject matter relates to compounds having a chemical structure shown in Formula I or II, particularly Formulas IA, IB, and IC, as defined herein. In yet a further aspect, the disclosed subject matter relates to a method for the treatment of an oncological disease in a patient. For example, disclosed herein is a method of administering an effective amount of a compound or composition disclosed herein to a patient having an oncological disorder, eg, having melanoma, and in need thereof. Is done. Also disclosed are methods of using the disclosed compounds to inhibit or kill tumor cells, inhibit HDAC6, and enhance tumor inflammatory responses.

Additional advantages of the disclosed subject matter will be set forth in part in the following description and drawings, and in part will be apparent from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

The materials, compounds, compositions, articles, and methods described herein are more readily understood by reference to the following detailed description of specific embodiments of the disclosed subject matter, and the examples and drawings contained therein. can do.

Prior to disclosing and describing the present materials, compounds, compositions and methods, the embodiments described below are limited to that particular synthesis method or particular reagent, as the particular synthesis method or particular reagent may of course vary. It should be understood that not. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Also, various publications are referenced throughout this specification. The entire disclosures of these publications are incorporated herein by reference to more fully describe the state of the art to which the disclosed problems relate. The disclosed references are also incorporated herein by reference, individually and in particular, with respect to the materials contained therein, which are stated in the text, on which the reference depends.

General Definitions In this specification and in the claims that follow, reference will be made to a number of terms that are defined to have the following meanings.

Throughout the description and claims, the word “comprise” and other forms of this word, “comprising” and “comprises” include, but are not limited to It is meant to be non-limiting, and is not intended to exclude other adjuncts, components, integers or steps, for example.

As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes a mixture of two or more such compositions, and a reference to “a compound” includes a mixture of two or more such compounds, and refers to “a drug” A reference includes a mixture of two or more such agents, as well as other cases.

“Optional” or “optionally” means that the event or situation described below may or may not occur, and the description includes when the event or situation occurs and when it does not occur.

Ranges may be expressed herein as from “about” one particular value and / or to another “about” one particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, where a value is expressed as an approximation by using the antecedent “about”, this particular value will be understood to form another aspect. It will be further understood that the endpoints of each range are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, each of which is also disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed, the value “below”, “above that value”, and possible ranges between values are also disclosed, as will be appreciated by those skilled in the art. For example, if the value “10” is disclosed, “10 or less” and “10 or more” are also disclosed. It will also be understood that throughout the specification, data is provided in a number of different formats, which represents the end point and start point, and the range of any combination of data points. For example, if a specific data point “10” and a specific data point “15” are disclosed, more than 10 and 15, more than 10 and 15, less than 10 and 15, less than 10, and less than 10, and It is understood that a value equal to 15 and between 10 and 15 is disclosed. It is also understood that each singular number between two specific singular numbers is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13 and 14 are also disclosed.

As used herein, “subject” means an individual. Therefore, “subjects” include domesticated animals (eg, cats, dogs, etc.), livestock (eg, cattle, horses, pigs, sheep, goats, etc.), laboratory animals (eg, mice, rabbits, rats, guinea pigs). Etc.), and birds. “Subject” may also include mammals such as primates or humans.

Other forms of the word such as “reducing” or “reducing” or “reducing” mean a reduction in an event or characteristic (eg, tumor growth). It is understood that this is typically related to a certain criterion or expected value, in other words, it is relative, but it is not always necessary to refer to the criterion or relative value. For example, “reduce tumor growth” means to reduce the growth rate relative to a reference or control.

Other forms of the word, such as “prevent” or “preventing” or “prevention”, stop certain events or features and stabilize or delay the development or progression of certain events or features Or minimizing the chance that a particular event or feature will occur. Preventing is typically more absolute than, for example, reducing and therefore does not require comparison with a control. As used herein, things may be reduced but not prevented, but things that are reduced may also be prevented. Similarly, things may be prevented but not reduced, but things that are prevented may also be reduced. It is understood that when reducing or preventing is used, the use of the other word is also explicitly disclosed unless specifically indicated otherwise.

Other forms of the word such as “treat” or “treated” or “treatment” administer the composition or perform the method to reduce a particular characteristic or event (eg, tumor growth or survival) Means prevention, inhibition or elimination. The term “control” is used synonymously with the term “treat”.

The term “anti-cancer” refers to the ability to treat or control cell proliferation and / or tumor growth at any concentration.

Throughout this specification, it will be understood that the identifiers “first” and “second” are used to merely distinguish the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to imply any particular order, amount, priority or importance to a component or process modified with these terms.

Chemical Definitions As used herein, the term “substituted” is intended to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and non-aromatic substituents of organic compounds. Exemplary substituents include those described below, for example. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For the purposes of the present invention, a heteroatom such as nitrogen has a hydrogen substituent and / or any permissible substituent of the organic compounds described herein that satisfy the valence of the heteroatom. Also good. This disclosure is not intended in any way to be limited to the permissible substituents of organic compounds. Also, the term “substituted” or “substituted” refers to compounds in which such substitution is in accordance with the permissible valence of the substituent atom and substituent, and the substitution is stable, eg, rearranged, cyclized. , Including implicit conditions that result in compounds that do not spontaneously undergo transformations such as exclusion.

“Z ¹ ”, “Z ² ,” “Z ³ ” and “Z ⁴ ” are used herein as generic symbols to represent various specific substituents. These symbols may be any substituents not limited to those disclosed herein, and if they are defined as being given substituents when present, they are otherwise Can be defined as several other substituents.

The term “aliphatic” as used herein refers to a non-aromatic hydrocarbon group, including branched and unbranched alkyl, alkenyl or alkynyl groups.

The term “alkyl” as used herein refers to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, A branched or unbranched saturated hydrocarbon group consisting of 1 to 24 carbon atoms, such as eicosyl and tetracosyl. Alkyl groups can also be substituted or unsubstituted. Alkyl groups are alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, as described below. It may be substituted with one or more groups including but not limited to sulfooxo, sulfonyl, sulfone, sulfoxide, or thiol.

Throughout the specification, “alkyl” is generally used to refer to both unsubstituted and substituted alkyl groups; however, a substituted alkyl group also refers to a specific substituent on an alkyl group by Special reference is made herein. For example, the term “alkyl halide” specifically refers to an alkyl group that is substituted with one or more halides, such as fluorine, chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “alkylamino” specifically refers to an alkyl group substituted with one or more amino groups as described below. When "alkyl" is used in some cases and specific terms such as "alkyl alcohol" are used in other cases, the term "alkyl" is implied to not refer to a specific term such as "alkyl alcohol". Does not mean to do.

This convention is also used for the other groups described herein. That is, terms such as “cycloalkyl” refer to both unsubstituted and substituted cycloalkyl moieties, where the substituted moiety is specified in more detail herein; for example, a particular substituted cycloalkyl is, for example, “ May be referred to as "alkylcycloalkyl". Similarly, substituted alkoxy may be specifically referred to as, for example, “halogenated alkoxy”, and a particular substituted alkenyl may be referred to as, for example, “alkenyl alcohol”. Again, the use of a generic term such as “cycloalkyl” and the use of a particular term such as “alkylcycloalkyl” does not imply that the generic term does not include a particular term.

The term “alkoxy” as used herein is an alkyl group attached through a single terminal ether linkage; that is, an “alkoxy” group can be defined as —OZ ¹ , where Z ¹ is as defined above. Alkyl.

The term “alkenyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (Z ¹ Z ² ) C═C (Z ³ Z ⁴ ) are intended to include both E and Z isomers. It can be assumed that there is an asymmetric alkene in the structural formula, or can be clearly indicated by the bond symbol C = C. Alkenyl groups are alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, as described below It may be substituted with one or more groups including but not limited to sulfooxo, sulfonyl, sulfone, sulfoxide, or thiol.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. Alkynyl groups are alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, as described below It may be substituted with one or more groups including but not limited to sulfooxo, sulfonyl, sulfone, sulfoxide, or thiol.

The term “aryl” as used herein is a group containing any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term “heteroaryl” is defined as a group comprising an aromatic group having at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to nitrogen, oxygen, sulfur and phosphorus. The term “non-heteroaryl” included in the term “aryl” defines a group that contains an aromatic group that does not contain a heteroatom. An aryl or heteroaryl group may be substituted or unsubstituted. Aryl or heteroaryl groups are alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, as described herein. May be substituted with one or more groups including, but not limited to, nitro, silyl, sulfooxo, sulfonyl, sulfone, sulfoxide, or thiol. The term “biaryl” is a specific type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups bonded to each other through a fused ring structure, as in naphthalene, or attached via one or more carbon-carbon bonds, as in biphenyl.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. The term “heterocycloalkyl” is a cycloalkyl group as defined above, wherein at least one of the ring carbon atoms is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur or phosphorus. Cycloalkyl groups and heterocycloalkyl groups may be substituted or unsubstituted. Cycloalkyl and heterocycloalkyl groups are alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, as described herein. It may be substituted with one or more groups including but not limited to nitro, silyl, sulfooxo, sulfonyl, sulfone, sulfoxide, or thiol.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one double bond, ie, C═C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above wherein at least one of the ring carbon atoms is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur or phosphorus. In the meaning of the term “cycloalkenyl”. Cycloalkenyl groups and heterocycloalkenyl groups may be substituted or unsubstituted. Cycloalkenyl groups and heterocycloalkenyl groups are alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, as described herein. It may be substituted with one or more groups including but not limited to nitro, silyl, sulfooxo, sulfonyl, sulfone, sulfoxide, or thiol.

The term “cyclic group” is used to refer to either an aryl group, a non-aryl group (ie, a cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl group) or both. Cyclic groups have one or more ring systems that may be substituted or unsubstituted. Cyclic groups may include one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.

The term “aldehyde” as used herein is represented by the formula —C (O) H. Throughout this specification “C (O)” or “CO” is an abbreviation for C═O, also referred to herein as “carbonyl”.

The term “amine” or “amino” as used herein is represented by the formula —NZ ¹ Z ² , where Z ¹ and Z ² are each hydrogen, alkyl as described above, halogenated alkyl, alkenyl, alkynyl, aryl. Or a substituent described herein, such as a heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group. “Amido” is —C (O) NZ ¹ Z ² .

The term “carboxylic acid” as used herein is represented by the formula —C (O) OH. As used herein, a “carboxylate” or “carboxyl” group is represented by the formula —C (O) O 2 ^— .

The term “ester” as used herein is represented by the formula —OC (O) Z ¹ or —C (O) OZ ¹ , where Z ¹ is alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, as described above. It may be a heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group.

The term “ether” as used herein is represented by the formula Z ¹ OZ ² , wherein Z ¹ and Z ² are independently alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclo, as described above. It may be an alkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group.

The term “ketone” as used herein is represented by the formula Z ¹ (O) Z ² , where Z ¹ and Z ² are independently alkyl, alkyl halide, alkenyl, alkynyl, aryl, hetero, as described above. It may be an aryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group.

As used herein, the term “halide” or “halogen” refers to fluorine, chlorine, bromine and iodine.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “nitro” as used herein is represented by the formula —NO ₂ .

The term “silyl” as used herein is represented by the formula —SiZ ¹ Z ² Z ³ , where Z ¹ , Z ² and Z ³ are independently hydrogen, alkyl as described above, halogenated alkyl, alkoxy, It may be an alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group.

The term “sulfonyl” refers herein to a sulfooxo group represented by the formula —S (O) ₂ Z ¹ , where Z ¹ is hydrogen, alkyl as described above, alkyl halide, alkenyl, alkynyl, aryl, hetero, It may be an aryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group.

The term “sulfonylamino” or “sulfonamide” as used herein is represented by the formula —S (O) ₂ NH—.

The term “thiol” as used herein is represented by the formula —SH.

The term “thio” as used herein is represented by the formula —S—.

As used herein, “R ¹ ”, “R ² ”, “R ³ ”, “R ⁿ ” and the like (where n is an integer) independently represent one or more of the groups listed above. You may own it. For example, when R ¹ is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group may optionally be substituted with a hydroxyl group, an alkoxy group, an amine group, an alkyl group, a halide, and the like. Depending on the group selected, the first group may be incorporated into the second group, or alternatively, the first group may be suspended (ie, attached to) the second group. For example, the expression “an alkyl group containing an amino group” may be incorporated into the backbone of the alkyl group. Alternatively, the amino group may be attached to the backbone of the alkyl group. The nature of the group selected will determine whether the first group is embedded or attached to the second group.

Unless stated to the contrary, formulas having chemical bonds, which are indicated only by solid lines and not by wedges or dashed lines, represent each possible isomer, for example each enantiomer, diastereomer, and meso compound, and racemic or scalemic mixtures. A mixture of isomers such as is envisaged.

Reference will now be made in detail to certain aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.

Compounds A variety of HDAC6 inhibitors have been studied (Butler et al., “Rational Design and Simple Chemistry Yield a Superior, Neuroprotective HDAC6 Inhibitor 108 CJ A13, C”). Kalin et al., “Second-Generation Histone Deacetylase 6 Inhibitors Enhancement the Immunosuppressive Effects of Foxp3 + T-Regulatory Cells”, JM 39 (JM 39). Characteristic of these agents is the presence of a benzyl linker containing a “cap-linker-zinc linking group” system built into a reference inhibitor. There is a report disclosing HDACi that does not have a zinc bonding group (ZBG) (Vickers et al, “Discovery of HDAC Inhibitors That Lack an Active Site Zn ²⁺ -Binding Functional Group M AC L (6): 505-508). These drugs possess moderate activity against class 1 enzymes.

The compounds disclosed herein maintain a ZBG, most preferably a hydroxamic acid group. In addition, the disclosed compounds contain certain urea-based cap groups incorporated into the benzylhydroxamic acid backbone, resulting in potent and selective HDAC6 inhibitors with anti-melanoma activity in vitro. Accordingly, the present specification includes formula I:

Or a pharmaceutically acceptable salt or hydrate thereof,
Wherein A is aryl, heteroaryl, or C ₁ -C ₈ alkyl, any of which is optionally acetyl, C ₁ -C ₅ alkyl, amino, —NR ⁶ R ⁷ , —C (O ) NR ⁶ R ⁷ , C ₁ -C ₄ alkoxy, C ₁ -C ₄ alkylhydroxy, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, heteroaryl, halo, hydroxy, thiol, cyano Or substituted with one or more groups selected from nitro; R ¹ and R ² are hydrogen, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkenyl, C ₁ -C ₈ alkynyl, C ₁ -C _{8 haloalkyl,} C 5 -C _{6 cycloalkyl,} C 5 -C _{6 heterocycloalkyl,} C 1 -C ₃ alkylaryl, _aryl, C 1 -C ₃ alkylheteroaryl, or Are independently selected from heteroaryl, either of which optionally _acetyl, C 1 -C ₅ alkyl, ^{amino, -NR 6 R 7, -C (} O) NR 6 R 7, C 1 ~C 4 alkoxy Substituted with C ₁ -C ₄ alkylhydroxy, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, heteroaryl, carbonyl, halo, hydroxy, thiol, cyano, or nitro; or R ¹ And R ² are joined together to form an alkylene bridge containing 2 atoms to form a 5-membered ring with the —NC (O) N— moiety, wherein A is Or a 5-membered ring optionally substituted with R ¹ ′, R ² ′, R ¹ ″, and R ² ″, which are independently hydrogen, or _C 1 _{~C 8} alkyl , _C 5 -C _{6 cycloalkyl,} C 5 -C _{6 heterocycloalkyl,} a C 1 -C ₃ alkylaryl, _aryl, C 1 -C ₃ alkylheteroaryl, or heteroaryl, either of which optionally Substituted with amino, aryl, C ₁ -C ₄ alkoxy, halo, or hydroxy; or R ¹ ′ and R ¹ ″ together or R ² ″ and R ² ′ together That is, = O); or R ¹ ′ and R ² ′ are absent and R ¹ ″ and R ² ″ together form a condensed phenyl group; R ⁶ and R ⁷ are Independently H, C ₁ -C ₄ alkyl, or joined together to form an alkylene bridge containing 4 or 5 atoms, linked to form a 5 or 6 membered ring with nitrogen .

In some examples, when R ¹ and R ² are both hydrogen, A is not hydroxyphenyl.

In certain instances, A is optionally C ₁ -C ₅ alkyl, amino, alkoxy, alkyl hydroxy, halo, hydroxy, or phenyl substituted with thiol, pyridyl, oxazolidyl, or a pyrimidyl. In the example of Naobetsu, A is phenyl, or _C 1 -C _{5 alkyl,} C 1 -C ₄ alkoxyl, or may be a phenyl substituted with halo. In the example of Naobetsu, A is pyridyl, or _C 1 -C _{5 alkyl,} C 1 -C ₄ alkoxyl, or may be a pyridyl substituted with halo. In preferred examples, A is phenyl or methoxyl substituted phenyl or halo substituted phenyl. In a further example, A may be phenyl. In still further examples, A may be phenyl substituted with one or more methoxyl, ethoxyl, or propoxyl groups, for example, A is one at the ortho-, para-, or meta-position. It may be phenyl substituted with a methoxyl group. In the most preferred example, A may be phenyl substituted with a methoxyl group in the ortho-position. Still further, A may be phenyl substituted with one or more halo groups, for example, A is one halo in the ortho-, para-, or meta-position (eg, Cl, Br, or F) Phenyl having a group may be used. In another example, A may be phenyl having one or more carboxylic acid groups or alkyl ester groups (eg, acetyl groups). A _can be a C 1 -C ₈ alkyl group. In yet another example, A may be phenyl substituted with one or more C ₁ -C ₄ alkyl groups. A is phenyl substituted with one methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, s-butyl, i-butyl group in the ortho-, para-, or meta-position. It may be. A may be phenyl substituted with one or more NH ₂ or N (C ₁ -C ₄ ) ₂ groups.

In another example, A may be an n-propyl, i-propyl, n-butyl, t-butyl, s-butyl, i-butyl, n-pentyl, i-pentyl, or s-pentyl group. .

In particular examples, R ¹ may be hydrogen, C ₁ -C ₈ alkyl, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, or heteroaryl, any of these It is optionally _C 1 -C ₃ alkyl, _{^{amino, -NR 6 R 7, C 1}} ~C 4 _alkoxy, C 1 -C ₄ alkyl hydroxy, carbonyl, hydroxy, substituted thiol, or cyano. In particular examples, R ¹ may be C ₁ -C ₈ alkyl, such as C ₁ -C ₄ alkyl. In another example, R ¹ is optionally acetyl, NH ₂ , N (C ₁ -C ₄ ) ₂ C ₁ -C ₄ alkoxy, C ₁ -C ₄ C ₅ -C ₆ heterocycloalkyl, carbonyl, halo, Or it may be C ₁ -C ₈ alkyl substituted with hydroxy. In a preferred example, R ¹ is hydrogen.

In particular examples, R ² can be hydrogen, C ₁ -C ₈ alkyl, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, or heteroaryl, any of these It is optionally _C 1 -C ₅ alkyl, _{^{amino, -NR 6 R 7, C 1}} ~C 4 _alkoxy, C 1 -C ₄ alkyl hydroxy, carbonyl, hydroxy, substituted thiol, or cyano. In a particular embodiment, ^{R 2} _is, C 1 -C ₅ alkyl, or methoxy, amino, ^-NR 6 ^{R 7,} alkyl hydroxy, carbonyl, hydroxy, be a _C 1 -C ₅ alkyl substituted with cyano Good. In another example, R ² may be C ₁ -C ₄ alkyl substituted with a heteroaryl such as imidazole or indole. In another example, R ² may be C ₁ -C ₄ alkyl substituted with phenyl, hydroxy substituted phenyl, methoxy substituted phenyl, halo substituted phenyl, or amino substituted phenyl.

In a further example, the disclosed compounds have the formula IA

Or a pharmaceutically acceptable salt or hydrate thereof,
Wherein R ² is as described herein; each W is independently CH or N independently of the others; R ⁵ is hydrogen, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkenyl, C ₁ -C ₈ alkynyl, C ₁ -C ₈ haloalkyl, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, or heteroaryl, any of which are optionally _acetyl, C 1 -C ₅ alkyl, ^{amino, -NR 6 R 7, -C (} O) NR 6 R 7, C 1 ~C 4 _alkoxy, C 1 -C ₄ alkyl _hydroxy, C 5 -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, heteroaryl, carbonyl, halo, hydroxy, thiol, substituted cyano, or nitro.

In a further example, the disclosed compounds have the formula IB

Or a pharmaceutically acceptable salt or hydrate thereof,
Wherein R ¹ is as described herein; each W is independently CH or N independently of the others; R ⁵ is hydrogen, C ₁ -C ₈ alkyl, C ₁ -C _8. Alkenyl, C ₁ -C ₈ alkynyl, C ₁ -C ₈ haloalkyl, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, or heteroaryl, any of which is optionally acetyl , _C 1 -C ₅ alkyl, ^{amino, -NR 6 R 7, -C (} O) NR 6 R 7, C 1 ~C 4 _alkoxy, C 1 -C ₄ alkyl _hydroxy, C 5 -C ₆ cycloalkyl alkyl, C ₅ -C ₆ heterocycloalkyl, aryl, heteroaryl, carbonyl, halo, hydroxy, thiol, substituted cyano, or nitro.

The disclosed compound may be selective HDAC6i. A homology model (Butler et al, J Am Chem Soc 2010, 132 (31): 10842-1084) shows that the entrance to the binding site is wider and shallower in HDAC6 compared to HDAC1. This model also shows lipophilic cavities. Accordingly, the disclosed compounds may include certain branch elements incorporated into the arylurea cap group primarily at A, R ¹ and / or R ² , which branch elements have access to this side cavity. Resulting in both improved efficacy and selectivity and better interaction with the surface of HDAC6.

Access to this side cavity may in one aspect be achieved through substitution of the nitrogen atom of the urea linker, ie R ² of formula I, proximal to the benzyl linker. The synthesis of these branched acyclic ureas is accomplished as outlined in Scheme 1. As an example, various amines undergo reductive amination with methyl 4-formylbenzoate 2 to form the desired secondary amines 3a-h. Subsequently, the branched urea esters 4a-h are obtained by reacting 3a-h with a suitable isocyanate. Aryl isocyanates are shown in Scheme 1; however, other isocyanates may be used to vary the “A” group of formula I (eg, heteroaryl or alkyl). This chemistry produces a series of ureas with a branched substitution (R ² ) on the nitrogen proximal to the benzyl linker. Hydroxamic acid groups are introduced using hydroxylamine under basic conditions to provide hydroxamic acids 5a-h.

Scheme 1: Synthesis of proximal N-substituted hydroxamic acids

Reagents and ^{_{conditions: (a) R 2 -NH 2}} , NaCNBH 3, rt, 5% AcOH / DCM, 16h; (b) aryl -NCO, DCM, rt, 16h; (c) 50 _wt% NH 2 OH, NaOH , THF / MeOH (1: 1), 0 ° C. to rt, 30 min.

In another embodiment, access to this side cavity may be achieved through substitution of the nitrogen atom of the urea linker, ie, R ¹ of Formula I, distal to the benzyl linker. The synthesis of these branched acyclic ureas is accomplished as outlined in Scheme 2. As an example, a copper-mediated Buchwald coupling reaction was used to assemble anilines 6a, b from iodobenzene, since these intermediates are not commercially available (Kwon et al., “Copper-catalyzed). "coupling of alkylamines and aryl ioides": An efficient system even in an air atmosphere "Org Lett 2002, 4 (4): 581-584). A triphosgene chemistry is performed to convert methyl 4- (aminomethyl) benzoate to the corresponding isocyanate, which undergoes reaction with secondary amines 6a-c to provide the penultimate ester 7a-c. To do. Final conversion to hydroxamic acid is accomplished as in Scheme 1 to complete the synthesis of 8a-c.

Scheme 2: Synthesis of distal N-substituted hydroxamic acids

Reagents and conditions: (a) R ¹ —NH ₂ , CuI, K ₃ PO ₄ , ethylene glycol, iPrOH, 80 ° C., 18 h; (b) i. Triphosgene, sat. aq bicarbonate / DCM (1: 1), 0 ° C., 30 min; ii. Methyl 4- (aminomethyl) benzoate _{-HCl, Et 3 N, DCM,} rt, 16h; (c) 50 _{wt% NH 2 OH, NaOH, THF} / MeOH (1: 1), 0 ℃ ~rt, 30 minutes.

Specific compounds according to formula I are as follows:

Or a pharmaceutically acceptable salt or hydrate thereof.

In yet another aspect, access to the side cavities of HDAC6 is also via the linkage of the distal and proximal nitrogen atoms of the urea linker via an alkylene bridge, ie, connecting R ¹ and R ² together, It may be achieved by providing a 5-membered ring with the -NC (O) N- moiety of formula I. Thus, in a further example, the disclosed compounds have the formula IC

You may have
Where A is hydrogen, or A is aryl, heteroaryl, or C ₁ -C ₈ alkyl, any of which is optionally acetyl, C ₁ -C ₅ alkyl, amino, —NR ^{6 R 7, -C (O)} NR 6 R 7, C 1 ~C 4 _alkoxy, C 1 -C ₄ alkyl _hydroxy, C 5 -C _{6 cycloalkyl,} C 5 -C ₆ heterocycloalkyl, aryl, heteroaryl Substituted with one or more groups selected from halo, hydroxy, thiol, cyano, or nitro, wherein R ⁶ and R ⁷ are as defined above;
R ¹ , R ² , R ¹ ″ and R ² ″ are independently hydrogen, or C ₁ -C ₈ alkyl, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, C ₁ -C ₃ alkylaryl, aryl, C ₁ -C ₃ alkylheteroaryl, or heteroaryl, any of which are optionally substituted with amino, aryl, C ₁ -C ₄ alkoxy, halo, or hydroxy Or R ¹ ′ and R ¹ ″ together or R ² ″ and R ² ′ together form a carbonyl (ie, ═O); or R ¹ ′ and R ² ′ are Absent, R ¹ ″ and R ² ″ together form a fused phenyl group.

In certain examples, R ² ′ and R ² ″ are both methyl. In another example, ^{R 2} 'is hydrogen, R ^2' 'is methyl, ethyl, propyl, i- propyl, n- butyl, i- butyl, benzyl, tosyl, _{hydroxyphenyl,} C 1 -C ₄ alkoxyphenyl Or aminophenyl. In another example, R ² ′ and R ² ″ may form a carboxyl (ie, ═O) group, or are pharmaceutically acceptable salts or hydrates thereof.

In certain examples, R ¹ ′ and R ¹ ″ are both methyl. In another example, R ¹ ′ is hydrogen and R ¹ ″ is methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, benzyl, tosyl, hydroxyphenyl, C ₁ -C ₄ alkoxy It is phenyl or aminophenyl. In another example, R ¹ ′ and R ¹ ″ form a carboxyl (ie, ═O) group.

  The synthesis of these cyclic ureas is accomplished as outlined in Scheme 3.

Scheme 3: Synthesis of cyclic urea hydroxamic acid

Reagents and conditions: (a) i. aq NaOH, ii. H + 60 ° C. 1 h; (b) KOtBu, DMF, 0 ° C. to RT; (c) aqNH ₂ OH, NaOH, THF / MeOH; (d) triphosgene, Et ₃ N.

Specific examples of compounds of formula IC are as follows:

Or a pharmaceutically acceptable salt or hydrate thereof.

In one aspect, the disclosed compounds comprise a branched aryl or alkylurea cap group or a cyclic urea cap group introduced into a reference HDACi platform. The introduction of branching elements, in particular the introduction of the urea motif into the proximal nitrogen atom, shows excellent selectivity for HDAC6 over the full panel of HDACs, and compared to histone proteins, α-tubulin It has led to the discovery of potent inhibitors that can induce selective hyperacetylation. The SAR that has been developed up to this point shows that the branched urea skeleton confers substantial benefits on the desired biochemical activity. These compounds were also screened in cell lines and found that both 5g and 5h can inhibit the growth of the B16 melanoma cell line.

Also disclosed are hydroxamate compounds lacking a urea motif. Such compounds have the formula II

Or a pharmaceutically acceptable salt or hydrate thereof,
Wherein R ² is as defined above and R ⁸ is acetyl, C ₁ -C ₅ alkyloxycarbonyl, carbobenzyloxy, methoxybenzylcarbonyl, benzoyl, benzyl, methoxybenzyl, dimethoxybenzyl, methoxyphenyl, C ₁ -C ₅ alkyl carbamate, or arylsulfonyl, ie, R ⁹ (SO ₂ ), where R ⁹ is aryl optionally substituted with C ₁ -C ₅ alkyl, amino, methoxyl, halo, or hydroxy. . In preferred examples, R ⁸ may be C ₁ -C ₅ alkyloxycarbonyl or arylsulfonyl. Specific examples of compounds of formula II are as follows:

Or a pharmaceutically acceptable salt or hydrate thereof.

  Also disclosed are pharmaceutically acceptable salts and hydrates of the disclosed compounds. Pharmaceutically acceptable salts include salts of the disclosed compounds prepared with acids or bases, depending on the particular substituents found on the compound. Administration of the compound as a salt may be appropriate under conditions where the compound disclosed herein is sufficiently basic or acidic to form a stable non-toxic acid or base salt. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, or magnesium salt. Examples of physiologically acceptable acid addition salts include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, carbonic acid, sulfuric acid, and acetic acid, propionic acid, benzoic acid, succinic acid, fumaric acid, mandelic acid, sulphate. Examples thereof include organic acids such as acid, citric acid, tartaric acid, malonic acid, ascorbic acid, α-ketoglutaric acid, α-glycophosphoric acid, maleic acid, tosylic acid, and methanesulfonic acid. Therefore, the present specification includes hydrochloride, nitrate, phosphate, carbonate, bicarbonate, sulfate, acetate, propionate, benzoate, succinate, fumarate, mandelate Oxalate, citrate, tartrate, malonate, ascorbate, α-ketoglutarate, α-glycophosphate, maleate, tosylate, and mesylate. Pharmaceutically acceptable salts of the compounds can be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid. Physiologically acceptable anions can be obtained. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

Methods of Use Further provided herein is a method of treating or preventing cancer in a subject, the method comprising administering to the subject an effective amount of a compound or composition disclosed herein. In addition, the method may further comprise administering an effective amount of ionizing radiation to the subject.

Also provided herein are methods for killing tumor cells. The method includes contacting a tumor cell with an effective amount of a compound or composition disclosed herein. The method may further comprise administering a second compound or composition (eg, an anticancer agent) to the subject, or administering an effective amount of ionizing radiation to the subject.

Also provided herein is tumor radiation therapy, the method comprising contacting a tumor with an effective amount of a compound or composition disclosed herein and irradiating the tumor with an effective amount of ionizing radiation. including. Also provided herein are methods for treating inflammation in a subject, the method comprising administering to the subject an effective amount of a compound or composition disclosed herein. Optionally, the method may further comprise administering a second compound or composition (eg, an anti-inflammatory agent).

  The disclosed subject matter also relates to methods for treating a subject having an oncological disease or condition. In one embodiment, an effective amount of one or more compounds or compositions disclosed herein is administered to a subject having an oncological disorder and in need of such treatment. The disclosed methods may optionally include identifying a subject in need or may require treatment of an oncological disorder. The subject may be a human or primate (monkey, chimpanzee, ape, etc.), other mammals such as dogs, cats, cows, pigs or horses, or other animals with oncological diseases. Means of administering and formulating compounds for administration to a subject are known in the art, examples of which are described herein. Oncological diseases include anus, bile duct, bladder, bone, bone marrow, intestine (including colon and rectum), breast, eye, gallbladder, kidney, oral cavity, larynx, esophagus, stomach, testis, cervix, head , Cervix, ovary, lung, mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid, sputum, vulva, uterus, liver, muscle, pancreas, prostate, blood cells (including lymphocytes and other immune system cells) ), And brain cancers and / or tumors. Specific cancers that are expected to be treated include carcinoma, Karposi's sarcoma, melanoma, mesothelioma, soft tissue sarcoma, pancreatic cancer, lung cancer, leukemia (acute leukemia, acute myeloid, chronic lymphocytic, chronic Examples include myeloid and other), and lymphomas (Hodgkin and non-Hodgkin), and multiple myeloma.

Other examples of cancers that can be treated according to the methods disclosed herein include adrenocortical cancer, adrenocortical cancer, brain astrocytoma, basal cell cancer, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, bar Kit lymphoma, carcinoid tumor, central nervous system lymphoma, cervical cancer, chronic myeloproliferative disease, colon cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, gallbladder cancer, gastric (gastric ( (stomach)) cancer, gastrointestinal carcinoid tumor, germ cell tumor, glial cell type, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, hypothalamic and visual tract glial cell type, eyeball Internal melanoma, retinoblastoma, pancreatic islet cell carcinoma (endocrine pancreas), laryngeal cancer, lip and oral cancer, liver cancer, medulloblastoma, Merkel cell carcinoma, occult mycosis fungiides Squamous neck cancer, myelodysplastic syndrome, myeloid leukemia, nasal cavity and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, Paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, cell plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, Prostate cancer, rectal cancer, renal cell (kidney) cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing sarcoma, soft tissue sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, extratendiary primitive nerve Germ layer tumor, testicular cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, choriocarcinoma, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

Melanoma and Mantle Cell Lymphoma A preferred embodiment of the specification disclosed herein is a method of treating a subject with melanoma by administering an effective amount of a compound of Formula 1 or II. Melanoma is currently the fastest growing cancer according to the World Health Organization. Currently, there are few therapies that provide a significant increase in survival in metastatic melanoma. Immunotherapy is an attractive modality with potentially few side effects due to the antigenic specificity of adaptive immunity. The latest therapy approved by the FDA for the treatment of melanoma was the antibody to CTLA-4, a major regulator of T cell activity, ipilimumab; however, this therapy is moderate in overall survival Provide improvements.

To overcome the mechanism of tumor-mediated immune suppression, it is necessary to target multiple pathways. One strategy that has received attention is the use of histone deacetylase inhibitors (HDACi). Indeed, HDACi treatment has been shown to enhance the expression of immunologically related genes such as MHC and costimulatory molecules. Inhibition of IL-10 is a potent anti-inflammatory cytokine after treatment of macrophages with HDACi. However; most studies to date have used pan-HDACi, which inhibits all 11 zinc-dependent HDACs. Therefore, more selective use of HDACi is preferred to minimize side effects.

As shown herein, HDAC6 is a molecular target at least in melanoma. Both HDAC6 pharmacological and gene disruption in B16 mouse melanoma cells, using HDAC6-selective inhibitor (HDAC6i) and target shRNA (HDAC6KD), respectively, measured by propidium iodide staining for DNA content. It resulted in an inhibition of proliferation characterized by arrest. Furthermore, HDAC6i was used to provide improved expression of immunologically related receptors including MHC-I and MHC-II. In vivo, subcutaneous injection of HDAC6KD B16 cells into wild type mice resulted in delayed tumor growth compared to control cells. However, this effect was suppressed in experiments using SCID mice lacking T and B cells, suggesting an important immune component for tumor control in vivo.

The mechanism by which HDAC6 regulates tumor immunogenicity has not yet been clarified. One possible mechanism arises from the protein immunoprecipitation test, which shows that HDAC6 interacts with STAT3, which is an important survival and virulence factor in melanoma and also involved in immune tolerance, Indicates potential adjustment.

  Expressed HDAC6 was found by gene microarray analysis to be upregulated in tumor biopsies of most melanoma patients compared to normal skin. This observation was supported by a microarray of patient melanoma tissue, stained immunohistochemically.

Overall, HDAC6 inhibition is an attractive therapeutic target in melanoma and mantle cell lymphoma, both by slowing tumor growth and providing a more attractive immune target, and the development and use of selective HDAC6i Provide a rationale for

Inflammatory Response Tumor antigen-specific CD8 + T cells have previously been shown to be non-responsive in patients with melanoma (Lee et al., Nat. Med. 1999, 5: 677-85). T cells infiltrating the bone marrow of patients with multiple myeloma are also non-responsive (Noonan et al. Cancer Res. 65: 2026-34, 2005). The conclusion of these studies, along with several other reports in the literature, is that CD4 + T cells are tolerated against tumor antigens early in tumor progression. This presents a significant obstacle to the development of effective cancer immunotherapy.

The role of histone deacetylase (HDAC) in epigenetic regulation of inflammatory responses in APC is disclosed herein. HDACs are a group of enzymes that regulate chromatin structure and gene expression by removing acetyl groups from lysine residues on histones. HDACs are classified into four different classes shown in FIG.

HDAC is a target of histone deacetylase inhibitor (HDACi) as shown in FIG. HDACi is a structurally diverse compound that can target several HDACs. HDACi induces differentiation, cell cycle and growth arrest in cancer cells. There is a new role for HDACi as a modulator of inflammation and anti-tumor responses.

Pan-HDACi LAQ824 has previously been found to enhance the inflammatory response in macrophages through transcriptional regulation of IL-10. Pan-HDACI LAQ824 was also found to restore tolerant T cell responsiveness (Wang et al. J Immunol 2011, 186: 3986-96). The mechanism of pan-HDACI and related targets are difficult to elucidate considering their multiple effects. Understanding the expression and function of specific HDACs in APC can reveal novel targets that affect immune activation versus tolerance. The identified target is then susceptible to pharmacological inhibition with isoform-selective HDACi.

HDAC6 was found to affect IL-10 gene expression within APC, as shown in FIG. HDAC6 is a 131 kDa protein encoded on the X chromosome and is predominantly cytoplasmic; however, recent data suggests that HDAC6 may also be present in the nucleus. HDAC6 has tubulin deacetylase activity associated with cell motility and T cell / APC synapses. There are isoform-selective HDAC6 inhibitors available. FIG. 4 shows that HDAC6 gene or pharmacological disruption inhibits IL-10. FIG. 5 shows that HDAC6 gene disruption improves APC function. As shown in FIG. 6, the mechanism shown by CHIP analysis of the IL-10 gene promoter in macrophages is H3 and H4 acetylation; HDAC6 mobilization; and STAT3 and other transcription factors at several time points after LPS stimulation Including the combination.

FIG. 7 shows that HDAC6 knockdown results in reduced recruitment of the transcriptional activator STAT3 to the IL-10 gene promoter. The C-terminus of HDAC6 is required for interaction with HDAC11. FIG. 8 shows that disruption of STAT3 binding to the gene promoter resulted in decreased recruitment of HDAC6 and decreased IL-10 production. FIG. 9 shows that disruption of HDAC6 inhibits STAT3 phosphorylation.

FIG. 10 shows that there is increased expression of HDAC6 and IL-10 mRNA in human melanoma. FIG. 11 is a series of images showing HDAC6 expression in mouse and human melanoma cell lines. FIG. 12 is a series of images showing HDAC protein expression in melanoma. FIG. 13 is a series of images showing reduced proliferation and cell cycle arrest in melanoma cells lacking HDAC6. FIG. 14 is a series of images showing that melanoma cells lacking HDAC6 are more immunogenic.

As shown in FIGS. 14A and 14B, B16 cells treated with HDAC6i ST-2-92 showed higher expression of MHC-I and -II molecules compared to untreated B16 cells. Similar changes in MHC expression were observed in B16 cells in which HDAC6 was knocked down. Of note, delayed tumor growth was observed in C57BL16 mice challenged with B16-KDDAC6 cells in vivo (FIG. 14C). This slowing of tumor growth in KDHDAC6 melanoma cells may be a reflection of their reduced proliferation (FIGS. 12, 13) and / or their increased immunogenicity resulting in improved immune recognition and clearance. To address this question, C57BL16 SCID mice were challenged with either KDHDAC6 or WTB16 melanoma cells. Unlike immune competent mice in which a delay in KDDAC6 tumor growth was observed (FIG. 14C); such an effect was not observed in SCID mice challenged with the same KDDAC6 cells (FIG. 14D). These results suggest that the immunological effects induced by the destruction of HDAC6 in melanoma cells make these cells “deeper understanding” by the immune system.

FIG. 15 is a series of images showing that pharmacological inhibition of HDAC6 in melanoma cells resulted in cell cycle arrest and increased expression of MHC molecules. It has also been found that melanoma cells treated with HDAC6-specific inhibitors are better activators of T cells (CD4 and / or CD8). The procedure for this discovery is to load OVA-peptides into melanoma cells (treated with or without Tubastatin A), add OT-lor OT-II transgenic T cells (naïve or tolerized), and Determining the production of IL-2 and IFN-γ.

FIG. 16 is an image showing that Tubastatin A inhibits JAK2 / STAT3 phosphorylation in B16 mouse melanoma cells in vivo. FIG. 17 is a series of images showing that Tubastatin A enhances the antigen-specific CD4 + T cell response to vaccination in melanoma-supported mice. In vivo, there is an anti-melanoma effect after administration of Tubastatin A (alone or in combination with anti-CLTA4).

FIG. 18 shows that Tubastatin A, a selective HDAC6 inhibitor, reduced STAT3 phosphorylation and recruitment to the IL-10 gene promoter in APC. FIG. 19 shows phenotypic and functional changes within APC treated with Tubastatin A. FIG. 20 shows that Tubastatin A-treated APC is a better activator of naive T cells and restores response of response-invisible T cells. FIG. 21 is a graph showing the antitumor effect of Tubastatin A in vivo. FIG. 22 shows that Tubastatin A does not affect PEM. FIG. 23 is a series of images showing the immunological effect of Tubastatin A on macrophages. FIG. 24 is a series of images showing that Tubastatin A inhibits IL-10 transcription by disrupting the JAKISTAT3 pathway in macrophages. FIG. 25 is a graph showing that the inhibitory effect of Tubastatin A on IL-10 production is lost in the absence of HDAC6. FIG. 26 is a flowchart showing the experimental design of an in vitro antigen presentation test. FIG. 27 is a series of images showing that Tubastatin A-treated macrophages are better activators of naive T cells and restore function of response-invisible T cells. FIG. 28 is a series of images showing that treatment with Tubastatin A in vivo enhances the response of antigen-specific T cells to vaccination.

Experiments with Tubastatin A in the above APC show that treatment of macrophages with Tubastatin A increases the expression of costimulatory molecules and inhibits IL-10 production by these cells. Tubastatin A-treated macrophages are better activators of naïve T cells and restore response-insensitive T cell function in vitro. In vivo treatment with Tubastatin A enhances the response of antigen-specific T cells to vaccination. Mechanistically, Tubastatin A disrupts the JAKISTAT3 / IL-iO pathway and balances it to immunogenic macrophages rather than tolerogenic.

FIG. 29 is an image showing HDAC6 expression in human MCL. FIG. 30 is a series of images showing the destruction of HDAC6 in a human MCL cell line. FIG. 31 is an image showing the destruction of HDAC6 in mouse FC-muMCLI cells. FIG. 32 is an image showing the immunological effects of HDAC6 inhibition within MCL. FIG. 6 shows changes in MHC, costimulatory molecules and / or cytokine production in response to LPS or CpG +/− ST-3-06 or Tubastatin A. FIG. FIG. 33 is a series of images showing the antigen presenting function of FC-muMCLI cells treated with ST-3-06. FIG. 34 is a series of images showing the antigen presentation function of FC-muMCLI cells treated with Tubastatin A. FIG. 35 is an image showing the antitumor effect of Tubastatin A in vivo. The data shows that HDAC6 inhibition enhances the immunogenicity of MCL cells. HDAC6 is required for STAT3 activation within APC, and STAT3 reduces the immunogenicity of tumor cells.

  FIG. 36 is a series of images showing that HDAC6 disruption inhibits STAT3 phosphorylation within APC. The C-terminus of HDAC6 is required for interaction with HDAC11. (A) HDAC6 construct encoding proteins of different lengths and carrying the FLAG epitope. (B) HDAC6 constructs are overexpressed in HeLa cells and their expression was assessed by Western blot using anti-FLAG antibody, or (C) immunoprecipitated to assess their interaction with HDAC11 .

FIG. 37 is a series of images showing that ST-3-06 reduced STAT3 phosphorylation and recruitment to the IL-10 gene promoter in APC. Human MCL cells show improved expression of HDAC6. The destruction of HDAC6 in malignant B cells inhibits their proliferation and is associated with the induction of apoptosis. Pharmacological or gene disruption of HDAC6 in MCL cells enhances their antigen-presenting ability in vitro, resulting in better T cell activation and restoration of response-invisible T cell function. Treatment of MCL-supported mice with Tubastatin A in vivo is associated with a strong anti-tumor effect. Mechanistically, HDAC6 has been found to interact with STAT3 within APC. The rationale for using HDAC6-specific inhibitors alone or in combination with STAT3 inhibitors within MCL is disclosed herein.

Compositions, Formulations and Methods of Administration In vivo application of the disclosed compounds, and compositions containing them, can be accomplished by any suitable method and technique known now or in the future to those skilled in the art. For example, the disclosed compounds are formulated in a physiologically or pharmaceutically acceptable form and are any suitable known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. It may be administered by any route. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. Administration of the disclosed compounds or compositions may be a single dose, or continuous or individual intervals, as can be readily determined by one skilled in the art.

The compounds disclosed herein, and compositions containing them, can also be administered using liposome technology, delayed release capsules, implantable pumps, and biodegradable containers. These delivery methods can advantageously provide a uniform dosage over an extended period of time. The compounds may also be administered in their salt derivative form or crystalline form.

The compounds disclosed herein may be formulated according to known methods for preparing pharmaceutically acceptable compositions. The formulations are well known to those skilled in the art and are described in detail in a number of readily available sources. For example, E.I. W. Remington's Pharmaceutical Science by Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein may be formulated so that an effective amount of the compound is mixed with a suitable carrier to facilitate effective administration of the compound. The composition used may also be in a variety of forms. They include, for example, tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and injectable solutions, and solid, semi-solid, and liquid dosage forms such as sprays. The preferred dosage form depends on the intended mode of administration and therapeutic application. The composition preferably also includes conventional pharmaceutically acceptable carriers and diluents known to those skilled in the art. Examples of carriers or diluents used with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To administer their dosages for the desired therapeutic treatment, the compositions disclosed herein are advantageously about 0. 0, based on the weight of the total composition including the carrier or diluent. 1% to 99% by weight, in particular 1 to 15% by weight of one or more total target compounds may be included.

Formulations suitable for administration include, for example, antioxidants, buffers, bacteriostats, and aqueous sterile injectable solutions that may contain solutes that render the formulation isotonic and isotonic with the blood of the intended recipient; And aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. The formulation may be present in unit dose or multi-dose containers, such as sealed ampoules and vials, and in a sterile liquid carrier, such as freeze-dried, which requires only distilled water for injection prior to use. It may be stored in a (lyophilized) state. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, tablets, and the like. In addition to the ingredients specifically mentioned above, it should be understood that the compositions disclosed herein may include other agents customary in the art related to the type of formulation. .

The compounds disclosed herein, and compositions comprising them, can be delivered to cells by direct contact with the cells or via carrier means. Carrier means for delivering compounds and compositions to cells are known in the art and include, for example, encapsulating the composition within a liposome portion. Other means for delivering the compounds and compositions disclosed herein to cells include attaching the compounds to proteins or nucleic acids targeted for delivery to target cells. U.S. Pat. No. 6,960,648, and U.S. Patent Application Publication Nos. 2003032594 and 20020120100 can bind to other compositions that cross biological membranes. An amino acid sequence that enables migration is disclosed. US Patent Publication No. 20020035243 also describes a composition for transporting biological moieties across a cell membrane for intracellular delivery. The compounds may also be incorporated into polymers, examples of which in the case of intracranial tumors are poly (DL lactide-co-glycolide) polymers; poly [bis (p- Carboxyphenoxy) propane: sebacic acid] (as used in GLIADEL); chondroitin; chitin; and chitosan.

For the treatment of oncological diseases, the compounds disclosed herein may contain other anti-tumor or anti-cancer substances and / or radiation and / or photodynamic therapy and / or surgery to remove the tumor. In combination with physical treatment, it can be administered to a patient in need of treatment. These other substances or treatments can be given at the same time or at different times as the compounds disclosed herein. For example, the compounds disclosed herein include mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, adriamycin or DNA interventions such as bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, anti-angiogenic agents such as angiostatin, anti-estrogenic agents such as tamoxifen, and / or, for example, GLEEVEC (Novatis Pharmaceuticals Corporation) and HERCEPTIN, Inc.) with other anti-cancer drugs or antibodies, or immunotherapeutic drugs such as ipilimumab and bortezomib Can be used in combination. In another aspect, the disclosed compounds are co-administered with other HDAC inhibitors such as ACY-1215, Tubacin, Tubastatin A, ST-3-06, or ST-2-92.

In certain instances, the compounds and compositions disclosed herein may be desirably administered at one or more anatomical sites, for example, desirably in combination with a pharmaceutically acceptable carrier such as an inert diluent. It may be administered locally at the site of no cell growth (such as tumor site or benign skin growth, eg injection or topical application to tumor or skin growth). The compounds and compositions disclosed herein may be administered intravenously or orally, etc., optionally in combination with an inert diluent or, for oral delivery, a pharmaceutically acceptable carrier such as an edible edible carrier. May be administered systemically. The compounds and compositions may be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of the patient's diet. For oral therapeutic administration, the active compound is combined with one or more excipients and ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers It may be used in the form of an aerosol spray or the like.

Tablets, troches, pills, capsules and the like may also include: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; corn starch, potato starch, alginic acid and the like Tablet disintegrating agents; lubricants such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose, or aspartame, or flavoring agents such as peppermint, wintergreen oil, or cherry flavor may be added. Where the unit dosage form is a capsule, the capsule may contain, in addition to materials of the above type, a liquid carrier such as vegetable oil or polyethylene glycol. There may be various other materials that otherwise alter the physical form of the coating or solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar or the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release formulations and devices.

The compounds and compositions disclosed herein, including pharmaceutically acceptable salts or hydrates thereof, may be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts may be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof, and oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion are sterile aqueous solutions or dispersions containing the active ingredients that are adapted for immediate preparation of injectable or injectable sterile solutions or dispersions, optionally encapsulated in liposomes. Liquids or sterile powders may be included. The final dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle is a solvent or liquid dispersion medium comprising, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oil, non-toxic glyceryl ester, and suitable mixtures thereof. Also good. The proper fluidity can be maintained, for example, by the formation of liposomes or, in the case of dispersion, by the maintenance of the required particle size, or by the use of surfactants. Optionally, prevention of microbial activity can be brought about by various other antibacterial and antifungal agents such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by delaying absorption, for example, by including aluminum monostearate and gelatin.

Sterile injections are prepared by incorporating the required amount of a compound and / or agent disclosed herein in a suitable solvent, optionally together with various other ingredients listed above, followed by filter sterilization. Prepared. In the case of sterile powders for the preparation of sterile injectables, the preferred method of preparation is vacuum drying and lyophilization to provide the active ingredient and powders of any additional desired ingredients present in a previously sterile filtered solution Technology.

For topical administration, the compounds and agents disclosed herein may be applied as liquids or solids. In general, however, it may be desirable to administer topically to the skin as a composition that may be solid or liquid, in combination with a dermatologically acceptable carrier. The compounds and agents and compositions disclosed herein can be applied topically to the skin of a subject to reduce the size of malignant or benign growth (which can include complete removal) or to treat the site of infection. it can. The compounds and agents disclosed herein can be applied directly to the growth or infection site. The compound and drug are preferably applied to the site of growth or infection in formulations such as ointments, creams, lotions, solutions, tinctures and the like.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols, or water-alcohol / glycol blends in which the compound can be dissolved or dispersed at an effective level, optionally with the aid of a non-toxic surfactant. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resulting liquid composition may be applied, for example, from an absorbent pad, used to impregnate bandages and other dressings, or affected using a pump type or aerosol sprayer It may be sprayed over the area.

Applicable pastes, gels for direct application to the user's skin, using thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose, or modified inorganic materials with liquid carriers Ointments, soaps, etc. may be formed.

Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art.

Also disclosed are pharmaceutical compositions comprising a compound disclosed herein in combination with a pharmaceutically acceptable carrier. A pharmaceutical composition adapted for oral, topical or parenteral administration comprising an amount of a compound constitutes a preferred embodiment. The dose administered to a patient, particularly a human, must be sufficient to achieve a therapeutic response in the patient without lethal toxicity over an ideal time frame, and at an acceptable level It is preferable to cause the following side effects or pathological conditions. Persons skilled in the art will appreciate that the dosage varies according to the subject's condition (health), subject's weight, if present, the type of combination treatment, the frequency of treatment, the therapeutic ratio, and the severity and stage of the condition. You will recognize that it depends on.

Kits The disclosed subject matter also relates to packaged dosage formulations containing, in one or more containers, at least one inhibitor compound or composition disclosed herein, eg, any compound of Formulas I-II. . The packaged dosage formulation may optionally contain a pharmaceutically acceptable carrier or diluent in one or more containers. The packaged dosage formulation can optionally include other HDAC inhibitors, or immunotherapeutic agents such as ipilimumab, in addition to the inhibitor compounds or compositions disclosed herein.

Depending on the disease or disease state being treated, a suitable dose may be an amount that will reduce the proliferation or growth of the target cells. In the context of cancer, a suitable dose is one that will result in a concentration of the active agent that is known to achieve the desired response in cancerous tissue such as a malignant tumor. Preferred dosages are those that result in maximal inhibition of cancer cell growth without having unmanageable side effects. Administration of the compound and / or drug may be continuous or discrete intervals, as can be readily determined by one skilled in the art.

To administer their dosages for the desired therapeutic treatment, in some embodiments, the pharmaceutical compositions disclosed herein are based on the weight of the total composition including the carrier or diluent. About 0.1% to 45% by weight, in particular 1 to 15% by weight of one or more total target compounds. Illustratively, the dosage levels of active ingredient administered are: intravenous, 0.01 to about 20 mg / kg; intraperitoneal, 0.01 to about 100 mg / kg; subcutaneous, 0.01 to about 100 mg / kg; Intramuscular, 0.01 to about 100 mg / kg; oral, 0.01 to about 200 mg / kg, preferably about 1 to 100 mg / kg; intranasal instillation, 0.01 to about 20 mg / kg; It may be from 01 to about 20 mg / kg animal (body) weight.

Also disclosed are kits that contain a composition comprising a compound disclosed herein in one or more containers. The disclosed kit may optionally include a pharmaceutically acceptable carrier and / or diluent. In one embodiment, the kit includes one or more other components, appendages, or adjuvants described herein. In another embodiment, the kit may include one or more anti-cancer agents, etc. as described herein. In one embodiment, the kit includes instructions or packaging material that describes how to administer the compound or composition of the kit. The container of the kit may be of any suitable material, such as glass, plastic, metal, etc., and may be of any suitable size, shape, or configuration. In one embodiment, the compounds and / or agents disclosed herein are provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, the compounds and / or agents disclosed herein are provided in the kit as a liquid or solution. In one embodiment, the kit includes an ampoule or syringe containing the compounds and / or agents disclosed herein in liquid or solution form.

The following examples are set forth below to illustrate the methods, compositions, and results according to the disclosed subject matter. These examples are not intended to include all aspects of the subject matter disclosed herein, but are intended to illustrate exemplary methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

Efforts have been made to ensure accuracy with respect to numbers (eg, amounts, temperature, etc.) but some errors and deviations should be taken into account. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Celsius or ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions that can be used to optimize the purity and yield of the products obtained from the described process, such as component concentration, temperature, pressure, and other reaction ranges and conditions. To do. Only ideal and routine experimentation will be required to optimize these process conditions.

¹ H and ¹³ C spectra were obtained on a Bruker spectrometer using TMS as an internal reference. The following abbreviations for multiplicity were used: s = singlet, d = doublet, t = triplet, m = multiplet, dd = double doublet, br = wide. The reaction was monitored by TLC using pre-coated silica gel plates (Merck silica gel 60 F ₂₅₄ , thickness 250 μm) and visualized under UV light. LRMS experiments were performed using an Agilent 1946A LC-MSD with MeCN spiked with 0.1% formic acid and H ₂ O as the mobile phase. HRMS determination was performed with a Shimadzu IT-TOF instrument using MeCN spiked with 0.1% formic acid and H ₂ O as mobile phase. Flash chromatography was accomplished using an automated CombiFlash R _f system from Teledyne ISCO and a prepacked silica gel cartridge according to the recommended load capacity.

Preparatory HPLC was used for purification of all final compounds using a Shimadzu preparative liquid chromatograph with the following specifications: Column: ACE 5AQ with a particle size of 5 μm (150 × 21.2 mm) . How _{1-25~100% MeOH / H 2 O,} 30 min; 100% MeOH, 5 _{minutes; 100~25% MeOH / H 2 O} , 4 min. How _{2-8~100% MeOH / H 2 O,} 30 min; 100% MeOH, 5 _{minutes; 100~8% MeOH / H 2 O} , 4 min. METHOD 3-0% MeOH, 5 _{minutes; 0~100% MeOH / H 2 O} , 25 min; 100% MeOH, 5 _{minutes; 100~0% MeOH / H 2 O} , 4 min. Flow rate—monitored at 17 mL / min, 254 and 280 nm. Both solvents were spiked with 0.05% TFA. Analytical HPLC was performed using an Agilent 1100 series instrument with the following specifications: Column: Luna 5C18 (2) 100A (150 × 4.60 mm) 5 μm particle size; gradient −10-100% MeOH / H ₂ O, 18 minutes, 100% MeOH, 3 minutes; 100-10% MeOH / H ₂ O, 3 minutes; 10% MeOH / H ₂ O, 5 minutes. Both solvents were spiked with 0.05% TFA. The purity of all test compounds was> 95% as determined by analytical HPLC.

Synthesis of compounds: see scheme 1 methyl 4-(((3- (dimethylamino) propyl) amino) methyl) benzoate (3a)
The synthesis of 3a is a typical general procedure A: A round bottom flask containing methyl 4-formylbenzoate (328 mg, 2 mmol) and 3-dimethylaminopropylamine (0.252 mL, 2 mmol) is added to 5% AcOH. In DCM (10 mL). After 5 minutes, NaCNBH ₃ (126 mg, 2 mmol) was added in portions and the resulting mixture was stirred overnight at room temperature under Ar atmosphere. The reaction was quenched with 1N NaOH (10 mL) and the aqueous layer was extracted with DCM (3 × 10 mL). The combined organic extracts were washed with brine, dried over sodium sulfate, concentrated under vacuum and purified via flash chromatography to give the product as a waxy solid (313 mg, 63%). . ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.98 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 3.89 (s, 3H), 3 .84 (s, 2H), 2.79 (brs, 1H), 2.68 (t, J = 6.8 Hz, 2H), 2.35 (t, J = 6.8 Hz, 2H), 2.23 (S, 6H), 1.70 (quant, J = 6.8 Hz, 2H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.96, 145.27, 129.71, 128.87, 127.95, 58.04, 53.42, 51.99, 47.87, 45.36, 27.41. LRMS ESI: [M + H] ⁺ = 251.1.

Methyl 4-(((3-hydroxypropyl) amino) methyl) benzoate (3b)
Made according to General Procedure A to give a waxy solid (89 mg, 29%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.00 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 3.91 (s, 3H), 3 .90 (s, 2H), 3.78 (wide, 4H), 2.93 (t, J = 5.6 Hz, 2H), 1.78 to 1.73 (m, 2H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.79, 143.16, 129.77, 129.44, 128.26, 63.55, 53.14, 52.10, 48.88, 30.18. LRMS ESI: [M + H] ⁺ = 224.2.

Methyl 4-(((2- (1H-indol-3-yl) ethyl) amino) methyl) benzoate (3c)
Made according to General Procedure A to give a waxy solid (446 mg, 72%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.28 (brs, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 7.6 Hz, 1H), 7 .36 to 7.34 (m, 3H), 7.21 (t, J = 7.6 Hz, 1H), 7.13 (t, J = 7.6 Hz, 1H), 7.00 (s, 1H) 3.92 (s, 3H), 3.87 (s, 2H), 3.02 to 2.98 (m, 4H), 1.68 (brs, 1H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 167.06, 145.66, 136.34, 129.60, 128.62, 127.85, 127.31, 122.02, 121.89, 119.13, 118.74, 113.47, 111.15, 53.36, 51.97, 49.28, 25.64. LRMS ESI: [M + H] ⁺ = 309.1.

Methyl 4-(((4-hydroxyphenethyl) amino) methyl) benzoate (3d)
Made according to General Procedure A to give a waxy solid (130 mg, 46%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.97 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.01 (d, J = 8. 4 Hz, 2H), 6.71 (d, J = 8.4 Hz, 2H), 3.90 (s, 3H), 3.86 (s, 2H), 2.86 (t, J = 6.8 Hz, 2H), 2.76 (t, J = 6.8 Hz, 2H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 167.07, 154.60, 145.02, 131.05129.78, 129.75, 128.91, 128.04, 115.55, 53.29, 52. 08, 50.37, 35.02. LRMS ESI: [M + H] ⁺ = 268.1.

Methyl 4-(((3-methoxypropyl) amino) methyl) benzoate (3e)
Made according to General Procedure A to give a colorless oil (127 mg, 54%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.94 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 3.86 (s, 3H), 3 .80 (s, 2H), 3.41 (t, J = 6.4 Hz, 2H), 3.28 (s, 3H), 2.67 (t, J = 6.4 Hz, 2H), 1.72 (M, 2H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.88, 145.78, 129.54, 128.58, 127.76, 71.17, 58.48, 53.50, 51.85, 46.72, 29.83. LRMS ESI: [M + H] ⁺ = 238.2.

Methyl 4-(((2-methoxyethyl) amino) methyl) benzoate (3f)
Made according to general procedure A to give a colorless oil (29 mg, 13%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.97 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 3.88 (s, 3H), 3 .84 (s, 2H), 3.49 (t, J = 5.2 Hz, 2H), 3.33 (s, 3H), 2.77 (t, J = 5.2 Hz, 2H), 1.88 (Br, 1H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.98, 145.58, 129.65, 128.74, 127.90, 71.85, 58.74, 53.47, 51.94, 48.69. LRMS ESI: [M + H] ⁺ = 224.2.

Methyl 4-((butylamino) methyl) benzoate (3 g)
Made according to general procedure A to give a colorless oil (150 mg, 68%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.9 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 3.91 (s, 3H), 3 .84 (s, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.49 (m, 2H), 1.34 (m, 3H), 0.91 (t, J = 7) .2Hz, 3H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 167.00, 145.89, 129.65, 128.70, 127.87, 53.63, 51.95, 49.14, 32.14, 20.38, 13.93. LRMS ESI: [M + H] ⁺ = 222.1.

Methyl 4-((phenethylamino) methyl) benzoate (3h)
Made according to general procedure A to give a colorless oil (203 mg, 75%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.01 (m, 2H), 7.35 (m, 4H), 7.22 (m, 3H), 3.93 (s, 3H), 3.88 ( s, 2H), 2.89 (m, 4H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.97, 145.66, 139.81, 129.65, 128.65, 128.43, 127.82, 126.15, 53.38, 51.96, 50.46, 36.29. LRMS ESI: [M + H] ⁺ = 270.1.

Methyl 4-((1- (3- (dimethylamino) propyl) -3- (2-methoxyphenyl) ureido) methyl) -benzoate (4a)
The synthesis of 4a is a typical general procedure B. A solution of 3a (99 mg, 0.395 mmol) in DCM (5 mL) was added to the appropriate isocyanate (0.053 mL, 0.395 mmol) at room temperature under Ar atmosphere and the resulting solution was stirred overnight. The reaction was quenched with saturated bicarbonate (10 mL) and extracted with DCM (3 × 10 mL). The combined organics were washed with brine (15 mL), dried over sodium sulfate, concentrated in vacuo and purified via flash chromatography to give the urea ester as a waxy solid (156 mg, 98%). It was. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.64 (brs, 1H), 8.18 (d, J = 7.2 Hz, 1H), 7.99 (d, J = 8.0 Hz, 2H), 7 .40 (d, J = 8.0 Hz, 2H), 6.97 to 6.94 (m, 2H), 6.84 (d, J = 7.2 Hz, 1H), 4.64 (s, 2H) 3.91 (s, 3H), 3.81 (s, 3H), 3.42 (t, J = 6.0 Hz, 2H), 2.34 (t, J = 6.0 Hz, 2H), 2 .20 (s, 6H), 1.74 (quant, J = 6.0 Hz, 2H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.85, 156.64, 148.86, 143.75, 129.77, 129.43, 129.02, 127.53, 122.25, 120.94, 120.36, 109.92, 55.52, 54.60, 51.96, 49.19, 44.64, 44.18, 24.98. LRMS ESI: [M + H] ⁺ = 400.2.

Methyl 4-((1- (3-hydroxypropyl) -3- (2-methoxyphenyl) ureido) methyl) benzoate (4b)
Made according to General Procedure B to give a waxy solid (113 mg, 74%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.07 to 8.03 (m, 3H), 7.39 (d, J = 8.0 Hz, 2H), 7.20 (brs, 1H), 6.93 -6.90 (m, 2H), 6.77-6.47 (m, 1H), 4.59 (s, 2H), 3.91 (s, 3H), 3.67-3.62 (m) 7H), 1.80 to 1.77 (m, 2H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.65, 156.31, 147.85, 142.24, 130.14, 129.70, 128.43, 126.77, 122.44, 121.04, 119.48, 109.88, 58.25, 55.53, 52.12, 50.41, 44.00, 30.32. LRMS ESI: [M + H] ⁺ = 373.2.

Methyl 4-((1- (2- (1H-indol-3-yl) ethyl) -3- (2-methoxyphenyl) ureido) methyl) -benzoate (4c)
Made according to General Procedure B to give a solid (200 mg, 95%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.23 to 8.21 (m, 1H), 8.15 (brs, 1H), 7.99 (d, J = 8.0 Hz, 2H), 7.60. (D, J = 7.6 Hz, 1H), 7.36-7.34 (m, 3H), 7.22-7.10 (m, 3H), 7.02 (s, 1H), 6.97 To 6.94 (m, 2H), 6.81 to 6.79 (m, 1H), 4.59 (s, 2H), 3.91 (s, 3H), 3.70 to 3.67 (m) 5H), 3.12 (t, J = 7.6 Hz, 2H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.83, 155.17, 147.60, 143.28, 136.29, 129.98, 129.34, 128.79, 127.30, 127.13, 122.18, 122.08, 121.15, 119.51, 118.96, 118.46, 112.53, 111.29, 109.29, 55.56, 52.10, 50.89, 48. 98. LRMS ESI: [M + H] ⁺ = 458.2.

Methyl 4-((1- (4-hydroxyphenethyl) -3- (2-methoxyphenyl) ureido) methyl) -benzoate (4d)
Made according to General Procedure B to give a solid (178 mg, 90%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.15 (d, J = 4.8 Hz, 1H), 8.01 (d, J = 7.6 Hz, 2H), 7.35 (d, J = 8. 0 Hz, 2H), 7.11 (s, 1H), 7.02 (d, J = 8.0 Hz, 2H), 6.95 to 6.93 (m, 2H), 6.80 to 6.77 ( m, 3H), 6.43 (brs, 1H), 4.53 (s, 2H), 3.91 (s, 3H), 3.73 (s, 3H), 3.56 (t, J = 6) .8 Hz, 2H), 2.87 (t, J = 6.8 Hz, 2H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.89, 155.25, 154.99, 147.69, 142.93, 130.04, 129.83, 129.76, 129.40, 128.50, 127.21, 122.35, 121.18, 119.10, 115.67, 109.78, 55.60, 52.15, 50.97, 50.33, 33.88. LRMS: [M + H] ⁺ = 435.2.

Methyl 4-((1- (3-methoxypropyl) -3-phenylureido) methyl) benzoate (4e)
Made according to General Procedure B to give a colorless oil (70 mg, 73%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.00 (d, J = 8.4 Hz, 2H), 7.84 (s, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7 .40 (d, J = 8.0 Hz, 2H), 7.30 (m, 2H), 7.02 (t, J = 7.6 Hz, 1H), 4.64 (s, 2H), 3.92 (S, 3H), 3.49 (t, J = 5.2 Hz, 2H), 3.44 (s, 3H), 3.415 (t, J = 6.4 Hz, 2H), 1.77 (m 2H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.87, 156.36, 143.76, 139.85, 129.88, 129.18, 128.81, 127.72, 122.45, 119.17, 68.16, 58.63, 52.05, 49.41, 43.05, 27.55. LRMS ESI: [M + H] ⁺ = 358.2.

Methyl 4-((1- (2-methoxyethyl) -3-phenylureido) methyl) benzoate (4f)
Made according to general procedure B to give a colorless oil (40 mg, 91%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.33 (br, 1H), 8.02 (d, J = 8.0 Hz, 2H), 7.35 (m, 6H), 7.02 (t, J = 7.2 Hz, 1H), 4.68 (s, 2H), 3.93 (s, 3H), 3.50 (s, 3H), 3.46 (s, 4H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.88, 157.07, 143.81, 139.37, 129.92, 129.28, 128.81, 127.73, 122.34, 119.15, 72.59, 59.28, 52.07, 50.90, 48.44. LRMS ESI: [M + H] ⁺ = 343.2.

Methyl 4-((1-butyl-3-phenylureido) methyl) benzoate (4 g)
Made according to general procedure B to give a colorless oil (59 mg, 98%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.04 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.27 (m, 4H), 7 .03 (t, J = 7.2 Hz, 1H), 6.32 (s, 1H), 4.65 (s, 2H), 3.93 (s, 3H), 3.36 (t, J = 7 .2 Hz, 2H), 1.64 (m, 2H), 1.37 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.74, 155.30, 143.11, 138.83, 130.14, 129.50, 128.85, 127.04, 123.16, 119.90, 52.12, 50.49, 47.74, 30.52, 20.18, 13.81. LRMS ESI: [M + H] ⁺ = 341.1.

Methyl 4-((1-phenethyl-3-phenylureido) methyl) benzoate (4h)
Made according to General Procedure B to give a colorless oil (113 mg, 92%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 (d, J = 8.4 Hz, 2H), 7.36 (m, 4H), 7.30 (d, J = 7.2 Hz, 1H), 7 .21 (m, 4H), 7.08 (d, J = 8.0 Hz, 2H), 6.99 (t, J = 7.2 Hz, 1H), 6.00 (s, 1H), 4.58 (S, 2H), 3.91 (s, 3H), 3.59 (t, J = 6.8 Hz, 2H), 2.90 (t, J = 6.8 Hz, 2H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.71, 155.61, 142.99, 138.86, 138.72, 130.06, 129.43, 129.00, 128.86, 128.65, 127.28, 126.92, 122.95, 119.83, 52.08, 50.38, 49.94, 34.74. LRMS ESI: [M + H] ⁺ = 389.2.

4-((1- (3- (dimethylamino) propyl) -3- (2-methoxyphenyl) ureido) methyl) -N-hydroxybenzamide (5a)
The synthesis of 5a is a typical general procedure C. Solid NaOH (125 mg, 3.12 mmol) was added aq. Dissolved in the solution (50 wt%, 1 mL). A solution of 4a (156 mg, 0.390 mmol) in THF / MeOH (1: 1, total 6 mL) was then added dropwise and the bilayer solution became homogeneous after complete addition. The resulting solution was stirred at room temperature for 30 minutes. The reaction was quenched with AcOH (0.223 mL, 3.90 mmol), concentrated in vacuo, and the crude product was purified via HPLC method 2 and neutralized with bicarbonate wash to give the title compound. (20 mg, 13%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.22 (brs, 1H), 9.02 (brs, 1H), 7.80-7.76 (m, 3H), 7.39 (d, J = 8.4 Hz, 2H), 7.00 to 6.94 (m, 2H), 6.88 to 6.84 (m, 2H), 4.62 (s, 2H), 3.72 (s, 3H) ), 3.43 to 3.40 (m, 2H), 2.82 (brs, 2H), 2.56 (s, 6H), 1.91 to 1.86 (m, 2H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 155.35, 149.62, 141.56, 131.69, 128.60, 127.24, 127.15, 127.02, 123.14, 121. 40, 120.23, 110.78, 55.62, 54.33, 49.32, 44.24, 42.76, 23.45. HRMS ESI: Calculated C ₂₁ H ₂₈ N ₄ O ₄ [M + H] ⁺ m / z = 401.2183; Found 401.2164.

N-hydroxy-4-((1- (3-hydroxypropyl) -3- (2-methoxyphenyl) ureido) methyl) -benzamide (5b)
Made according to General Procedure C and purified by Method 3 to give the title compound (95 mg, 84%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.19 (brs, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.76 to 7.71 (m, 3H), 7 .37 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 4.0 Hz, 2H), 6.88 to 6.85 (m, 1H), 4.58 (s, 2H) 3.74 (s, 3H), 3.48 (t, J = 5.6 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H), 1.73 to 1.69 (m, 2H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 164.10, 155.18, 149.08, 141.99, 131.56, 128.91, 127.18, 127.10, 126.93, 122. 61, 120.49, 120.29, 110.76, 57.59, 55.69, 49.28, 43.97, 30.61. HRMS ESI: Calculated C ₁₉ H ₂₃ N ₃ O ₅ [M + H] ⁺ m / z = 3744.1710; found 374.1693.

4-((1- (2- (1H-indol-3-yl) ethyl) -3- (2-methoxyphenyl) ureido) methyl) -N-hydroxybenzamide (5c)
Made according to General Procedure C and purified by Method 1 to give the title compound (62 mg, 57%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.21 (brs, 1H), 10.86 (s, 1H), 10.12 (brs, 1H), 7.86 (t, J = 7.6 Hz) 1H), 7.75 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 1H), 7.42-7.40 (m, 3H), 7.34 (D, J = 8.0 Hz, 1H), 7.18 (s, 1H), 7.07 (t, J = 7.2 Hz, 1H), 7.01 to 6.96 (m, 3H), 6 .90 to 6.86 (m, 1H), 4.64 (s, 2H), 3.71 (s, 3H), 3.62 (t, J = 7.6 Hz, 2H), 3.00 (t J = 7.6 Hz, 2H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 163.97, 158.37, 154.72, 148.88, 141.90, 136.21, 131.65, 128.70, 127.12, 122. 98, 122.65, 120.97, 120.35, 118.31, 118.18, 111.41, 111.11, 110.66, 55.66, 49.77, 48.47, 23.89. HRMS ESI: Calculated C ₂₆ H ₂₆ N ₄ O ₄ [M + H] ⁺ m / z = 459.2027; found 459.2030.

N-hydroxy-4-((1- (4-hydroxyphenethyl) -3- (2-methoxyphenyl) ureido) methyl) -benzamide (5d)
Made according to General Procedure C and purified by Method 2 to give the title compound (63 mg, 63%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.20 (brs, 1H), 9.20 (brs, 1H), 7.82 (d, J = 8.0 Hz, 2H), 7.74 (d , J = 8.0 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.37 (s, 1H), 7.04 (d, J = 8.4 Hz, 2H), 6 .98 (d, J = 8.0 Hz, 2H), 6.89 to 6.85 (m, 1H), 6.69 (d, J = 8.4 Hz, 2H), 4.57 (s, 2H) 3.75 (s, 3H), 3.49 (t, J = 7.6 Hz, 2H), 2.75 (t, J = 7.6 Hz, 2H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 164.00, 155.76, 154.62, 148.97, 141.88, 131.63, 129.63, 128.83, 127.12, 122. 72, 120.44, 120.33, 115.22, 110.69, 55.69, 49.68, 49.49, 33.21. HRMS ESI: Calculated C ₂₄ H ₂₅ N ₃ O ₅ [M + H] ⁺ m / z = 436. 1867; Found 436.1858.

N-hydroxy-4-((1- (3-methoxypropyl) -3-phenylureido) methyl) benzamide (5e)
Made according to General Procedure C and purified by Method 2 to give the title compound (48 mg, 76%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.19 (br, 1H), 8.36 (s, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.45 (d , J = 7.6 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.23 (m, 2H), 6.94 (t, J = 7.2 Hz, 1H), 4 .61 (s, 2H), 3.34 (m, 4H), 3.21 (s, 3H), 1.74 (m, 2H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 164.04, 155.30, 142.25, 140.42, 131.49, 128.31, 127.06, 121.87, 119.88, 69. 16, 57.88, 49.08, 43.56, 27.81. HRMS ESI: Calculated C ₁₉ H ₂₃ N ₃ O ₄ [M + H] ⁺ m / z = 358.1761; found 358.1785.

N-hydroxy-4-((1- (2-methoxyethyl) -3-phenylureido) methyl) benzamide (5f)
Made according to General Procedure C and purified by Method 2 to give the title compound (20 mg, 50%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.17 (s, 1H), 9.01 (br, 1H), 8.44 (s, 1H), 7.72 (d, J = 8.0 Hz) 2H), 7.42 (d, J = 7.6 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.23 (t, J = 7.6 Hz, 2H) H, 6.94 (t, J = 7.2 Hz, 1H), 4.64 (s, 2H), 3.49 (s, 4H), 3.28 (s, 3H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 164.06, 155.46, 142.17, 140.30, 131.43, 128.32, 126.97, 121.83, 119.64, 70. 88, 58.31, 49.82, 46.35. HRMS ESI: Calculated C ₁₈ H ₂₁ N ₃ O ₄ [M + H] ⁺ m / z = 3444.1605; found 344.1601.

4-((1-butyl-3-phenylureido) methyl) -N-hydroxybenzamide (5 g)
Made according to General Procedure C and purified by Method 2 to give the title compound (40 mg, 68%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.17 (br, 1H), 8.36 (s, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.46 (d , J = 7.6 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.22 (t, J = 7.6 Hz, 2H), 6.94 (t, J = 7. 2 Hz, 1 H), 4.62 (s, 2 H), 3.30 (m, 1 H), 1.48 (m, 2 H), 1.27 (m, 2 H), 0.86 (t, J = 7 .6Hz, 3H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 164.01, 155.22, 142.30, 140.45, 131.42, 128.19, 127.00, 126.96, 121.80, 120. 04, 49.01, 46.11, 29.64, 19.43, 13.77. HRMS ESI: Calculated C ₁₉ H ₂₃ N ₃ O ₃ [M + H] ⁺ m / z = 342.812; Found 342.1802.

N-hydroxy-4-((1-phenethyl-3-phenylureido) methyl) benzamide (5h)
Made according to General Procedure C and purified by Method 2 to give the title compound (71 mg, 63%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.15 (br, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H) 7.31 (d, J = 8.0 Hz, 2H), 7.24 (m, 7H), 6.95 (t, J = 7.2 Hz, 1H), 4.60 (s, 2H), 3 .54 (t, J = 7.6 Hz, 2H), 2.82 (t, J = 7.6 Hz, 2H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 164.04, 155.12, 142.12, 140.35, 139.04, 131.50, 128.77, 128.31, 128.20, 127. 09, 127.05, 126.16, 121.91, 120.13, 49.20, 48.04, 33.91. HRMS ESI: Calculated _{C 23 H 23 N 3 O 3} [M + H] + m / z = 390.1812; Found 390.1793.

Synthesis of compounds: see scheme 2 N-butylaniline (6a) was synthesized in a manner similar to that previously reported (Org Lett, 4,581) ¹¹ . Briefly, CuI (19 mg, 0.1 mmol) and freshly ground K ₃ PO ₄ (849 mg, 4 mmol) were placed in a sealed tube, followed by isopropanol (2 mL), ethylene glycol (0.222 mL, 4.. 0 mL), phenyl iodide (0.224 mL, 2.0 mmol) and n-butylamine (0.237 mL, 2.4 mmol) were added sequentially. The tube was then sealed and stirring was started at 80 ° C. for 18 h. After cooling to room temperature, the reaction was diluted with water: ethyl ether (1: 1, 10 mL). The aqueous layer was extracted with ether (3 × 5 mL), washed with brine (15 mL), dried over sodium sulfate and concentrated under vacuum. Purification via flash chromatography gave the title compound as a yellow oil (235 mg, 79%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.19 (m, 2H), 6.70 (t, J = 7.2 Hz, 1H), 6.61 (d, J = 8.4 Hz, 2H), 3 .60 (br, 1H), 3.12 (t, J = 7.2 Hz, 2H), 1.62 (m, 2H), 1.44 (m, 2H), 0.98 (t, J = 7) .2Hz, 3H). The spectrum is shown in Okano et al. , “Synthesis of secondary arylates through copper-mediated intermediate array”, Org Lett 2003, 5 (26): 4987-4990.

N- (3-methoxypropyl) aniline (6b)
Made according to the same procedure as 6a to give a pale yellow oil (282 mg, 85%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.18 (m, 2H), 6.69 (t, J = 7.2 Hz, 1H), 6.61 (d, J = 8.4 Hz, 2H), 3 .92 (br, 1H), 3.52 (t, J = 6.0 Hz, 2H), 3.60 (s, 3H), 3.23 (t, J = 6.8 Hz, 2H), 1.90 (M, 2H). The spectrum is shown in Guo et al. , Efficient Iron-Catalyzed N-Arylation of Aryl Halides with Amine ", Org Lett 2008, 10 (20): 4513-4516.

Methyl 4-((3-butyl-3-phenylureido) methyl) benzoate (7a)
Methyl 4- (aminomethyl) benzoate hydrochloride (101 mg, 0.5 mmol) was added to DCM: sat. Take up in a bilayer solution of bicarbonate (1: 1, 4 mL) and add triphosgene (49 mg, 0.17 mmol) at 0 ° C. After 30 minutes, the aqueous layer was extracted with DCM (3 × 5 mL), washed with brine (15 mL) and concentrated in vacuo. The crude isocyanate was taken up in DCM (2 mL), 6a (75 mg, 0.5 mmol) and Et ₃ N (0.209 mL, 1.5 mmol) were added and the resulting solution was stirred at room temperature overnight. The reaction is sat. Quenched with bicarbonate (5 mL) and extracted with DCM (3 × 5 mL). The combined organics were washed with brine (15 mL), dried over sodium sulfate and concentrated in vacuo. The crude material was purified via flash chromatography to give the title compound as an off-white waxy solid (93 mg, 55%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.95 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 7.6 Hz, 2H), 7.32 (m, 1H), 7 .25 (m, 4H), 4.45 (t, J = 5.6 Hz, 1H), 4.41 (d, J = 6.0 Hz, 2H), 3.89 (s, 3H), 3.70 (T, J = 7.6 Hz, 2H), 1.48 (m, 2H), 1.31 (m, 2H), 0.89 (t, 7.2 Hz, 3H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.89, 156.87, 145.14, 141.61, 130.08, 129.78, 128.85, 128.73, 127.81, 126.96, 52.01, 49.21, 44.25, 30.68, 19.92, 13.82. LRMS ESI: [M + H] ⁺ = 341.1.

Methyl 4-((3- (3-methoxypropyl) -3-phenylureido) methyl) benzoate (7b)
Prepared according to the procedure in 7a except that 6b was used as the secondary amine to give the title compound as an off-white waxy solid (65 mg, 36%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.96 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.33 (m, 1H), 7 .27 (m, 4H), 4.69 (br, 1H), 4.42 (6.0 Hz, 2H), 3.90 (s, 3H), 3.80 (t, J = 7.2 Hz, 2H ), 3.43 (t, J = 6.4 Hz, 2H), 3.27 (s, 3H), 1.83 (m, 2H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.88, 156.95, 145.05, 141.63, 130.10, 129.80, 128.90, 127.79, 127.00, 70.23, 58.54, 52.03, 46.86, 44.29, 28.82. LRMS ESI: [M + H] ⁺ = 357.1.

Methyl 4-((3-ethyl-3-phenylureido) methyl) benzoate (7c)
Prepared according to the procedure in 7a except using commercially available N-ethylaniline as the secondary amine to give the title compound as an off-white waxy solid (197 mg, 63%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.94 (d, J = 8.4 Hz, 2H), 7.24 (t, J = 7.6 Hz, 2H), 7.32 (t, J = 7. 2 Hz, 1 H), 7.26 (m, 4 H), 4.57 (br, 1 H), 4.41 (d, J = 5.6 Hz, 1 H), 3.88 (s, 3 H), 3.76 (Dd, J = 14, 7.2 Hz, 2H), 1.12 (t, J = 7.2 Hz, 3H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.82, 156.64, 145.09, 141.22, 130.03, 129.72, 128.79, 127.82, 126.92, 51.95, 44.17, 44.09, 13.82. LRMS ESI: [M + H] ⁺ = 313.1.

4-((3-Butyl-3-phenylureido) methyl) -N-hydroxybenzamide (8a)
Made according to General Procedure C and purified by Method 2, the title compound was obtained as an off-white solid (74 mg, 80%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.13 (br, 1H), 8.72 (br, 1H), 7.66 (d, J = 8.0 Hz, 2H), 7.43 (t , J = 7.6 Hz, 2H), 7.27 (m, 5H), 6.23 (t, J = 5.6 Hz, 1H), 4.20 (d, J = 5.6 Hz, 2H), 3 .57 (t, J = 6.8 Hz, 2H), 1.35 (m, 2H), 1.23 (m, 2H), 0.82 (t, J = 6.8 Hz, 3H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 164.17, 156.48, 144.50, 142.12, 130.129.55, 128.22, 126.67, 126.61, 48.44, 43.38, 30.21, 19.35, 13.72. HRMS ESI: Calculated C ₁₉ H ₂₃ N ₃ O ₃ [M + H] ⁺ m / z = 342.812; Found 342.1825.

N-hydroxy-4-((3- (3-methoxypropyl) -3-phenylureido) methyl) benzamide (8b)
Prepared according to General Procedure C and purified by Method 2 to give an off-white solid (59 mg, 91%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.14 (br, 1H), 7.66 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H) 7.27 (m, 5H), 6.27 (t, J = 6.0 Hz, 1H), 4.21 (d, J = 5.8 Hz, 2H), 3.62 (t, J = 7. 2 Hz, 2H), 3.28 (t, J = 6.4 Hz, 2H), 3.14 (s, 3H), 1.63 (m, 2H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 164.13, 156.51, 144.44, 142.19, 130.89, 129.57, 128.18, 126.70, 126.64, 69. 49, 57.80, 46.35, 43.39, 28.32. HRMS ESI: Calculated C ₁₉ H ₂₃ N ₃ O ₄ [M + H] ⁺ m / z = 358.1761; found 358.1749.

4-((3-Ethyl-3-phenylureido) methyl) -N-hydroxybenzamide (8c)
Made according to General Procedure C and purified by Method 2 to give an off-white solid (91 mg, 96%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.13 (br, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H) 7.26 (m, 5H), 6.30 (t, J = 6.0 Hz, 1H), 4.21 (d, J = 5.6 Hz, 2H), 3.61 (dd, J = 14, 7.2 Hz, 2H), 0.99 (t, J = 7.2 Hz, 3H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 164.21, 156.34, 144.54, 142.00, 130.92, 129.57, 128.32, 126.75, 126.67, 43. 70, 43.39, 13.76. HRMS ESI: Calculated C ₁₇ H ₁₉ N ₃ O ₃ [M + H] ⁺ m / z = 314.1499; found 314.1489.

HDAC Inhibition Assay Reaction Biology Corp. (Malvern, PA) performed HDAC inhibition assays using isolated human recombinant full length HDAC1 and -6 from the baculovirus expression system in Sf9 cells. The acetylated fluorescent peptide, RHKK _AC , derived from residues 379-382 of p53 was used as the substrate. Reaction buffer was made at a final concentration of 50 mM Tris-HCl pH 8.0, 127 mM NaCl, 2.7 mM KC1, 1 mM MgCl ₂ , 1 mg / mL BSA, and 1% DMSO. Compounds were fed into DMSO and enzyme mixture and pre-incubated for 5-10 minutes, then substrate was added and incubated at 30 ° C. for 2 h. Trichostatin A and a developing solution were added to quench the reaction and generate each fluorescence. Dose response curves were generated starting with 30 μΜ compounds using a 3-fold serial dilution and a 10-dose plot was generated. IC ₅₀ values are then generated from the resulting plots and the values represented are the mean ± standard deviation of duplicate tests.

Compound 1 was identified as possessing submicromolar HDAC inhibitory activity; however, it was selective for representative members of Zn ²⁺ -dependent class 1 and 2 (HDAC1 and HDAC6, respectively) It wasn't. It has been discovered that the activity and selectivity for HDAC6 can be improved by accessing a unique cavity on the HDAC6 surface. This was achieved by substitution on urea nitrogen. There is a short substrate channel on HDAC6 compared to HDAC1, and this feature has shown an excellent strategy conferring important isoform selectivity (Butler et al., J Am Chem Soc 2010, 132 ( 31): 10842-10846; Kalin et al., J Med Chem 2012, 55 (2): 639-651). By incorporating substituents on the urea motif, additional branched molecular surfaces can form valuable contacts due to slight differences in the HDAC6 surface, while the benzyl linker favors moving away from HDAC1 inhibition, more Give a short linker. A summary of the obtained HDAC1 and HDAC6 inhibition data is shown in Table 1.

Analogs based on 1 maintained the same 2-methoxyphenyl cap group, but included various substitutions on the proximal bound nitrogen of urea (5a-d). The introduction of branching elements at this position had a dramatic effect in reducing the activity of HDAC1. Interestingly, inhibition in HDAC6 was found to depend on the nature of this substitution. Both dimethylamino substitution, such as 5a, and 3-indoyl substitution, such as 5c, were more than 3 times less potent compared to compound 1 and proved harmful to HDAC6 inhibition. However, they maintained low micromolar inhibitory activity at HDAC1; however, activity against HDAC6 was in the submicromolar range only. Since the tertiary amine of 5a is protonated at physiological pH, a positive charge may be disadvantageous for proper target binding. Similarly, larger indole groups of 5c may simply exhibit too much steric bulk to be properly accommodated by the active site. However, the 3-hydroxypropyl derivative, 5b, and the 4-hydroxyphenylethyl derivative, 5d resulted in a significant increase in the inhibition of HDAC6. These substitutions had only a minor effect on HDAC1 activity. The 5b and 5d hydroxyl groups can function as H-bond acceptors or donors and have favorable interactions with major amino acid residues on the HDAC6 surface, thus improving binding affinity.

  A first series of compounds based on 1 maintained the 2-methoxy group within the arylurea cap. Since the oxidizability of phenol represents a significant hurdle for in vivo efficacy, structure-activity relationship (SAR) studies have been advanced by the synthesis of a series with phenyl caps using the same chemical structure. In order to show the effect of H-bond acceptors, the free hydroxyl moiety was masked. Capping the free hydroxyl with a methyl group yielded 5e, which was also found as a low nanomolar inhibitor with> 400-fold selectivity for HDAC6. Shortening of the 5f ethylene bridge did not significantly inhibit HDAC6 inhibition, but slightly increased activity against HDAC1 and ultimately reduced selectivity to HDAC6. The general trend established is that H-bond donors and large aromatic groups block activity, while smaller groups with H-bond acceptors favor selective HDAC6 activity. Interestingly, n-butyl 5g and phenethyl 5h were HDAC6i proficient in the low nanomolar range, with 5g having excellent selectivity over HDAC1 (600 times). These results negated the idea that certain H-bond interactions are required for activity.

Transferring the branch element to the distal urea nitrogen resulted in analogs 8a-c. The most powerful 8a in this series possessed the same n-butyl substitution as 5e. 8a is nanomolar HDAC6I, but 5 times lower in strength and more interestingly less selective than the proximally substituted homologue 5e. The methoxy variant 8b has dramatically reduced efficacy against HDAC6. Although the switch from alkyl to heteroalkyl on the proximal nitrogen resulted in equal potency inhibition on the distal nitrogen, this modification was detrimental to the development of more potent HDAC6I. Reducing the length of the alkyl branch at 8c also reduced HDAC6 inhibition. These data indicate that there is a specific need for inhibitors decorated with cap groups containing acyclic ureas, and that strong and selective inhibition is mostly due to urea substitution on the proximal nitrogen producing branched cap groups. Point out that it comes from.

Evaluating the disclosed compounds against other HDACi developed by others reveals that 5 g, termed “Nextastat A” is indeed a potent and selective HDAC6i. For example, comparing 5 g with another HDAC6i, Tubastatin A (Butler et al., J Am Chem Soc 2010, 132 (31): 10842-1084), while maintaining excellent selectivity for HDAC1, It becomes clear that the inhibition was improved. 5g also shows HDAC6 efficacy comparable to trichostatin A (TSA) (see Table 1). In addition, amino-benzamide ZBG has been incorporated into HDACi and its introduction reduces class 2 inhibition resulting in class 1 selectivity; this possesses anti-proliferative activity and recently started clinical trials It is symbolized by the HDACI MGCD0103 (Zhou et al., “Discovery of N- (2-aminophenyl) 4-4-pyridin-3-ylpyridine-2-ylamino) methyl benzamide (MGCD0103), deacetylase inhibitor ", J Med Chem 2008, 51 (14): 4072-4075). Compared to MGCD0103, 5 g results in a 30-fold reduction in activity in HDAC1.

Similar experiments were performed using compounds 10a, 10b and 11a, which are cyclic ureas of formula IC (Table 2).

Since HDAC isoforms are highly consistent with each other, obtaining selectivity is important to avoid off-target effects and is paramount to the development of the disclosed HDAC6i. Class 1 inhibition is well known to cause cytotoxicity associated with pan-selective HDACi; therefore, 5 g was screened against all 11 isoforms (Table 3). In similar class 1 and class 4 isoforms, 5 g showed low micromolar activity compared to the low nanomolar activity of HDAC6. In addition, 5 g showed a high level of selective inhibition over the related class 2 HDAC isoform members, in some cases reaching> 1000-fold selectivity. These data establish that 5 g, and similar analogs are potent and isoform selective HDAC6i.

Tubulin and Histone Acetylation Western Blot Assay 5 g was evaluated for its ability to induce α-tubulin hyperacetylation, a hallmark of HDAC6 inhibition, without increasing the level of acetylated histones. B16 melanoma cells were seeded at 10 ⁵ cells / well in 12-well plates and allowed to adhere overnight. A 50 mM stock of compound was then added into complete medium by serial dilution to the indicated concentrations. The cells were incubated for 24 hours under humidified conditions (37 ° C., 5% CO ₂ ). The wells were then washed with cold PBS and the cells were lysed in a buffer containing 10 mM Tris-HCl pH 8.0, 10% SDS, 4 mM urea, 100 mM DTT, and 1 × protease inhibitor (Roche). The cells were lysed on ice for 30 minutes and then sonicated for 8 minutes (8 cycles of 30 s / 30 s rest). Cells were then boiled with 6x gel loading buffer for 10 minutes, separated on a 4-15% gradient gel, and subsequently transferred onto a nitrocellulose membrane. Block membranes with 5% milk in PBS-T to detect specific antigens using antibodies to acetyl-H3 and H3 (Cell Signaling) and to acetyl-α-tubulin and α-tubulin (Sigma) did. Bands were detected by scanning the blot using both the 700 and 800 channels using the LI-COR Odyssey imaging system.

HDAC6 contains two catalytic domains. Its C-terminal domain is a functional domain for both synthetic and physiological substrates, while the N-terminal domain lacks enzymatic activity (Zou et al., “Characterization of the two catalytic domains in histone delacease 6”, Biochem Biophys Res Commun 2006, 341 (1): 45-50). Treatment with 5 g of low nanomolar on B16 murine melanoma cells resulted in a dose-dependent increase in acetyl α-tubulin levels without having a concomitant increase in histone H3 acetylation (FIG. 39), indicating binding to a second, catalytically active catalytic domain. Just before the 1 and 10 μM concentrations were used, an observable increase in histone H3 acetylation was found. This was expected because the 5 g biochemical IC ₅₀ for class 1 HDACs, which is responsible for histone acetylation, is in the micromolar range. There is a clear preference for 5 g of activity in the cellular environment corresponding to selective HDAC6 inhibition.

B16 Melanoma Cell Growth Inhibition Assay Compounds were evaluated in an MTS assay to determine the ability of selective HDAC6i to develop an antiproliferative effect on B16 mouse melanoma cells. B16 mouse melanoma cells were plated at 5 × 10 ³ / well in 96 well flat bottom plates. The next day, the media was replaced with media containing various concentrations of HDACi, or diluted in complete media to match DMSO vehicle concentrations and this was done in triplicate. Cells were incubated for 48 hours at 37 ° C. and 5% CO ₂ . The density of viable, metabolically active cells was quantified using a standard MTS assay (CellTiter 96 ™ _AQueous One, Promega, Madison, Wis.) According to the manufacturer's instructions. Briefly, 20 μL of reagent was added per well and incubated at 37 ° C. for 3 hours. Absorbance at 490 nM was measured spectrophotometrically using the background difference at 690 nM. All values were then normalized and expressed as a percentage of the medium control (100%).

Treatment of compounds for 48 h resulted in dose-dependent growth inhibition of oncogenic melanoma cells, which are summarized in Table 4. The general trend of inhibition of cell growth correlates with efficacy for HDAC6. However, the strong and selective 5b worked very poorly in cellular assays, probably because it does not have high polarity and efficient cell permeability. When compared to the most active compounds 5d and 5f-h in this whole cell assay, it becomes clear that the most selective HDAC6i has the greatest potency to inhibit cell growth. It should be noted that they also have higher cLogP values that may contribute to improved cell permeability. As illustrated for 5g (cLogP = 2.20), it shows that as cLogP is adjusted to a more optimal level, cell efficacy should be restored and an appropriate balance of physiochemical parameters should be maintained. ing.

Compared to pan-selective HDACi LBH589, 5 g is approximately 100 times less effective in inducing mouse B16 melanoma cell death. This decrease in potency is unlikely due to poor cell permeability as treatment of B16 cells with nanomolar doses of 5 g resulted in increased acetyl-tubulin levels as shown above (FIG. 39). ). In addition, both compounds possess similar cLogP values (2.64 vs. 2.20 for LBH589 and 5g, respectively). Rather, the effect of non-selective HDAC inhibition by LBH589 treatment may contribute to its effectiveness, particularly its class 1 activity. It is also interesting to note that 5g has increased efficacy against B16 cells compared to Tubastatin A (Table 4). There is no definitive explanation for the difference in cellular activity, but it may be due to an improvement in 5 g of HDAC6 activity. Although HDAC6-selective inhibitors have not had a role in cancer therapy to date, the data indicate that they have utility in this area. This study therefore constitutes the first report of a HDAC6 selective inhibitor possessing an antiproliferative effect on melanoma cells.

The materials and methods of the appended claims are not limited in scope by the specific materials and methods described herein, but are intended to be functionally equivalent, which are intended to exemplify several aspects of the claims. Any such materials and methods are included within the scope of the present disclosure. In addition to what is shown and described herein, various changes in materials and methods are intended to be included within the scope of the appended claims. Further, although only certain representative materials, methods, and aspects of those materials and methods have been described in detail, other materials and methods and various features of the materials and methods are not specifically cited. Are intended to be included within the scope of the appended claims. Thus, a process, element, component, or combination of components may be explicitly referred to herein, but all other combinations of steps, elements, components, or components may be referred to explicitly. included.

Claims (29)

Formula I:

Or a pharmaceutically acceptable salt or hydrate thereof,
Wherein A is aryl, heteroaryl, or C ₁ -C ₈ alkyl, any of which is optionally acetyl, C ₁ -C ₅ alkyl, amino, —NR ⁶ R ⁷ , —C (O ) NR ⁶ R ⁷ , C ₁ -C ₄ alkoxy, C ₁ -C ₄ alkylhydroxy, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, heteroaryl, halo, hydroxy, thiol, cyano Or substituted with one or more groups selected from nitro; R ¹ and R ² are hydrogen, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkenyl, C ₁ -C ₈ alkynyl, C ₁ -C _{8 haloalkyl,} C 5 -C _{6 cycloalkyl,} C 5 -C _{6 heterocycloalkyl,} C 1 -C ₃ alkylaryl, _aryl, C 1 -C ₃ alkylheteroaryl, or Are independently selected from heteroaryl, either of which optionally _acetyl, C 1 -C ₅ alkyl, ^{amino, -NR 6 R 7, -C (} O) NR 6 R 7, C 1 ~C 4 alkoxy Substituted with C ₁ -C ₄ alkylhydroxy, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, heteroaryl, carbonyl, halo, hydroxy, thiol, cyano, or nitro; or R ¹ And R ² are joined together to form an alkylene bridge containing 2 atoms to form a 5-membered ring with the —NC (O) N— moiety, wherein A is Or a 5-membered ring optionally substituted with R ¹ ′, R ² ′, R ¹ ″, and R ² ″, which are independently hydrogen, or _C 1 _{~C 8} alkyl , _C 5 -C _{6 cycloalkyl,} C 5 -C _{6 heterocycloalkyl,} a C 1 -C ₃ alkylaryl, _aryl, C 1 -C ₃ alkylheteroaryl, or heteroaryl, either of which optionally Substituted with amino, aryl, C ₁ -C ₄ alkoxy, halo, or hydroxy; or R ¹ ′ and R ¹ ″ together or R ² ″ and R ² ′ together That is, = O); or R ¹ ′ and R ² ′ are absent and R ¹ ″ and R ² ″ together form a condensed phenyl group;
R ⁶ and R ⁷ are independently H, C ₁ -C ₄ alkyl, or together form an alkylene bridge containing 4 or 5 atoms to form a 5 or 6 membered ring with nitrogen A compound that is linked as such. The compound of claim 1, wherein R ¹ and R ² are both hydrogen and A is not hydroxyphenyl. A is optionally C ₁ -C ₅ alkyl, amino, alkoxy, alkyl hydroxy, halo, hydroxy, or phenyl substituted with thiol, pyridyl, oxazolidyl, or pyrimidyl, A compound according to claim 1 or 2. 4. A compound according to any one of claims 1 to 3, wherein A is phenyl. A is one or more _C 1 -C _{5 alkyl,} C 1 -C ₄ alkoxyl, or halo is phenyl substituted with A compound according to any one of claims 1 to 4. 6. A compound according to any one of claims 1 to 5 wherein A is ortho-methoxyl substituted phenyl. A is pyridyl, or _C 1 -C _{5 alkyl,} C 1 -C ₄ alkoxyl, or pyridyl substituted with halo, A compound according to any one of claims 1 to 6. Any one of Claims 1-7 whose A is n-propyl, i-propyl, n-butyl, t-butyl, s-butyl, i-butyl, n-pentyl, i-pentyl, or s-pentyl group. A compound according to claim 1. R ¹ is hydrogen, C ₁ -C ₈ alkyl, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, or heteroaryl, any of which are optionally C ₁ -C ₃ alkyl, _{^{amino, -NR 6 R 7, C 1}} ~C 4 _alkoxy, C 1 -C ₄ alkyl hydroxy, carbonyl, hydroxy, substituted thiol, or cyano, according to any one of claims 1 to 8 Compound. The compound according to any one of claims 1 to 9, wherein R ¹ is C ₁ -C ₈ alkyl. R ¹ is optionally substituted with acetyl, NH ₂ , N (C ₁ -C ₄ ) ₂ C ₁ -C ₄ alkoxy, C ₁ -C ₄ C ₅ -C ₆ heterocycloalkyl, carbonyl, halo, or hydroxy and a _C 1 -C ₈ alkyl, a compound according to any one of claims 1 to 10. The compound according to any one of claims 1 to 11, wherein R ¹ is hydrogen. R ² is hydrogen, C ₁ -C ₈ alkyl, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, or heteroaryl, any of which are optionally C ₁ -C ₅ alkyl, _{^{amino, -NR 6 R 7, C 1}} ~C 4 _alkoxy, C 1 -C ₄ alkyl hydroxy, carbonyl, hydroxy, substituted thiol, or cyano, according to any one of claims 1 to 12 Compound. R ² is _C 1 -C ₅ alkyl, or methoxy, amino, ^-NR 6 ^{R 7,} alkyl hydroxy, carbonyl, hydroxy, _C 1 -C ₅ alkyl substituted with cyano, one of claims 1 to 13 A compound according to one paragraph. R ² is _C 1 -C ₄ alkyl substituted with heteroaryl, A compound according to any one of claims 1 to 14. R ² is phenyl, hydroxy-substituted phenyl, methoxy substituted phenyl, halo substituted phenyl, or C ₁ -C ₄ alkyl substituted with amino-substituted phenyl, A compound according to any one of claims 1 to 15. Formula IA:
Or a pharmaceutically acceptable salt or hydrate thereof,
In which each W is CH or N independently of the others; R ⁵ is hydrogen, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkenyl, C ₁ -C ₈ alkynyl, C ₁ -C ₈ haloalkyl, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, or heteroaryl, any of which are optionally acetyl, C ₁ -C ₅ alkyl, amino, —NR ^{6 R 7, -C (O) NR} 6 R 7, C 1 ~C 4 _alkoxy, C 1 -C ₄ alkyl _hydroxy, C 5 -C _{6 cycloalkyl,} C 5 -C ₆ heterocycloalkyl, aryl, heteroaryl, 17. A compound according to any one of claims 1-16, substituted with carbonyl, halo, hydroxy, thiol, cyano, or nitro. Formula IB:
Or a pharmaceutically acceptable salt or hydrate thereof,
In which each W is CH or N independently of the others; R ⁵ is hydrogen, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkenyl, C ₁ -C ₈ alkynyl, C ₁ -C ₈ haloalkyl, C ₅ -C ₆ cycloalkyl, C ₅ -C ₆ heterocycloalkyl, aryl, or heteroaryl, any of which are optionally acetyl, C ₁ -C ₅ alkyl, amino, —NR ^{6 R 7, -C (O) NR} 6 R 7, C 1 ~C 4 _alkoxy, C 1 -C ₄ alkyl _hydroxy, C 5 -C _{6 cycloalkyl,} C 5 -C ₆ heterocycloalkyl, aryl, heteroaryl, 18. A compound according to any one of claims 1 to 17 substituted with carbonyl, halo, hydroxy, thiol, cyano or nitro. Formula IC:

Or a pharmaceutically acceptable salt or hydrate thereof,
Wherein A is hydrogen, or A is aryl, heteroaryl, or C ₁ -C ₈ alkyl, any of which is optionally acetyl, C ₁ -C ₅ alkyl, amino, —NR ^{6 R 7, -C (O) NR} 6 R 7, C 1 ~C 4 _alkoxy, C 1 -C ₄ alkyl _hydroxy, C 5 -C _{6 cycloalkyl,} C 5 -C ₆ heterocycloalkyl, aryl, heteroaryl, Substituted with one or more groups selected from halo, hydroxy, thiol, cyano, or nitro, wherein R ⁶ and R ⁷ are as defined above; R ¹ ′, R ² ′, R ¹ '', and R ^{2 ''} is independently hydrogen, or _C 1 -C _{8 alkyl,} C 5 -C _{6 cycloalkyl,} C 5 -C _{6 heterocycloalkyl,} C 1 -C ₃ alkylaryl, Aryl, ₁ -C ₃ alkylheteroaryl, or heteroaryl, either of which optionally amino, _aryl, C 1 -C ₄ alkoxy, halo, or hydroxy substituted; or ^{R 1} 'and ^{R 1'} ' Together or R ² ″ and R ² ′ together form a carbonyl; or R ¹ ′ and R ² ′ are absent and R ¹ ″ and R ² ″ are together. The compound according to any one of claims 1 to 18, which forms a condensed phenyl group. R 1 ^'and R ^1' 'or R ^2' and R ^{2 ''} is methyl both A compound according to any one of claims 1 to 19. R ¹ ′ is hydrogen and R ¹ ″ is methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, benzyl, tosyl, hydroxyphenyl, C ₁ -C ₄ alkoxyphenyl, or aminophenyl Or R ² ′ is hydrogen and R ² ″ is methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, benzyl, tosyl, hydroxyphenyl, C ₁ -C ₄ alkoxyphenyl, Or the compound as described in any one of Claims 1-20 which is aminophenyl. R 1 ^'and R ^1' 'or R ^2' and R ^{2 ''} forms a carboxyl group, a compound according to any one of claims 1 to 21.

23. A compound according to any one of claims 1 to 22 selected from: A method of treating melanoma or mantle cell lymphoma in a subject, comprising administering to said subject a therapeutically effective amount of a histone deacetylase inhibitor. 25. The method of claim 24, wherein the inhibitor is a histone deacetylase 6 inhibitor. 26. The method of claim 24 or 25, wherein the inhibitor is Tubastatin A. 27. A method according to any one of claims 24-26, wherein the inhibitor is a compound according to any one of claims 1-23. 28. A method according to any one of claims 24-27, wherein the inhibitor is administered with one or more of ipilimumab, levlimide, velcade, vemurafenib, ST-3-06, ST-2-92, tubastatin A, tubacin. The method described. 29. A method according to any one of claims 24-28, wherein the inhibitor is combined with a STAT3 inhibitor.