LMO2 protein inhibitors

Abstract

The present invention relates to compounds of formula (I), wherein R, X, X, X, Q, R, R, and R are each as defined herein, that function as LMO2 active compounds. The invention also relates to processes for making these compounds, pharmaceutical compositions containing them, and their use in the treatment of proliferative diseases, such as cancer, and other diseases or conditions in which LMO2 activity is implicated. [Formula 1] JPEG2023538434000131.jpg48170

Description

[Technical Field]

[0001] This invention relates to specific pharmacologically active compounds that modulate the activity of T-cell leukemia chromosomal translocation proteins, specifically LIM domain-only protein 2 (LMO2). The compounds of this invention may be used to treat diseases or conditions at least partially mediated by inappropriate LMO2 activity, such as hyperproliferative diseases like cancer. The invention further relates to processes for producing these compounds, pharmaceuticals containing them, and their use as pharmaceutical compositions. [Background technology]

[0002] Tumor-associated chromosomal abnormalities that activate oncogenic proteins through gene activation or gene fusion are known to occur through chromosomal translocations, resulting in abnormal rearrangement of chromosomes. Recurring chromosomal translocations are abundant in leukemia / lymphoma, sarcoma, and carcinoma (Rabbitts, 2009) and represent a class of tumor-specific proteins that may be therapeutic targets. Generally, the products of chromosomal translocations are intracellular proteins that do not themselves possess an enzymatic active site but function in various cellular processes where protein-protein interactions (PPIs), such as transcription, are important. PPIs have a relatively large interaction surface containing several binding hotpots, but usually lack a distinct binding site (or pocket) (Scott et al., 2016). However, PPIs can be inhibited by macromolecules such as intracellular antibody fragments (e.g., single-chain fragment variable (scFv) (Cochet et al., 1998; Tanaka and Rabbittss, 2003; Visintin et al., 1999) or intracellular domain antibodies (iDAb) (Tanaka and Rabbittss, 2010; Tanaka et al., 2011; Tanaka et al., 2007)) and other antibody-like formats (Bery et al., 2019; Spencer-Smith et al., 2017). The advantage of intracellular antibody-based reagents is that they can leverage the natural properties of antibodies, such as their high affinity and specificity. Furthermore, its relatively rapid selection process, using methods such as intracellular antibody capture (Visintin et al., 1999), makes it possible to use it to study its effects on target diseases in relevant preclinical models (target validation) (Tanaka and Rabbitts, 2010; Tanaka et al., 2011; Tanaka et al., 2007).

[0003] While still being developed for the purpose of using intracellular antibodies as drugs themselves, known as macrodrugs (Tanaka and Rabbittss, 2008), the small size of the iDAb interaction surface with target antigens is being explored as a template for small molecule surrogates in a method called Abd technology (antibody-derived compound technology) (Quevedo et al., 2018). As reported by Quevedo et al., initial Abd selection was performed as a biochemical assay using competitive surface plasmon resonance (cSPR), where the interaction site of compounds from a fragment library with HRASG12V was evaluated by competition with HRAS-intracellular antibody dimers. From such in vitro selection methods, RAS-binding fragment hits developed by medicinal chemistry were obtained for nM interacting compounds. In vitro Abds exhibit favorable binding of intracellular antibodies to their targets (very high affinity, high on-rate constant (K)). on ) and a low dissociation rate constant (off-rate constant) (K off )), and also depend on selected compounds that have advantageous properties for intracellular uptake.

[0004] One intracellular protein produced from a chromosomal translocation is LIM domain-only protein 2 (LMO2), which is activated in T-cell acute lymphoblastic leukemia (T-ALL) by the chromosomal translocations t(11;14)(p13;q11) and t(7;11)(q35;p13) (Chambers and Rabbittss, 2015). LMO2 is overexpressed in over 50% of T-ALL patients (Ferrando and Look, 2003), and notably, it is not expressed in normal T cells (McCormack et al., 2003). Previously, to demonstrate that T-cell tumors do not proliferate when LMO2 is blocked (Tanaka et al., 2011), an intracellular VH, VH576 (hereinafter referred to as iDAb LMO2), which binds to LMO2, was used, and it was found that iDAb binds to LMO2, creating a stable structure that inhibits PPI with its natural partner (Sewell et al., 2014). Despite this, a compound that binds to the same interface of LMO2 as iDAb LMO2 is still needed to inhibit LMO2 PPI in cells and to regulate the activity of the T-cell leukemia chromosomal translocation protein, LMO2.

[0005] Considering the relevance of LMO2 proteins to the various physiological processes described above, LMO2 protein inhibitors, such as the compounds of the present invention, can be used in the treatment of various disease conditions in which LMO2 activity plays a role, is associated with inappropriate LMO2 activity, or where inhibition, regulation, or direct action of LMO2 protein-mediated signaling is desired. In summary, these studies suggest that selective inhibition of LMO2 proteins is a promising therapeutic approach, particularly in the treatment of hyperproliferative diseases such as cancer.

[0006] This invention was conceived with the above in mind. [Overview of the Initiative] [Means for solving the problem]

[0007] According to a first aspect of the present invention, compounds as defined herein, or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof are provided.

[0008] A further aspect of the present invention provides a pharmaceutical composition comprising a compound as defined herein, or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof, in a mixture with a pharmaceutically acceptable diluent or carrier.

[0009] A further aspect of the present invention provides a method for inhibiting LMO2 activity in vitro or in vivo, the method comprising contacting cells with an effective amount of a compound or a pharmaceutically acceptable salt thereof, a aqueous compound, or a solvated compound as defined herein.

[0010] A further aspect of the present invention provides a method for inhibiting LMO2 activity in vitro or in vivo, the method comprising contacting cells with an effective amount of a compound or a pharmaceutically acceptable salt thereof, a aqueous compound or a solvated compound, or a pharmaceutical composition as defined herein.

[0011] A further aspect of the present invention provides a method for treating a disease or disorder related to LMO2 activity in a patient requiring such treatment, the method comprising administering to the patient an effective amount of a compound as defined herein or a pharmaceutically acceptable salt thereof, a aqueous compound or a solvated compound thereof, or a pharmaceutical composition as defined herein.

[0012] A further aspect of the present invention provides a method for treating a proliferative disorder in a patient requiring such treatment, the method comprising administering to the patient an effective amount of a compound as defined herein or a pharmaceutically acceptable salt thereof, a aqueous compound or solvated compound thereof, or a pharmaceutical composition as defined herein.

[0013] A further aspect of the present invention provides a method for treating cancer in a patient requiring such treatment, the method comprising administering to the patient an effective amount of a compound as defined herein or an agent-acceptable salt thereof, a aqueous compound or solvated compound thereof, or a pharmaceutical composition as defined herein.

[0014] A further aspect of the present invention provides a compound as defined herein or a pharmaceutically acceptable salt thereof, a aqueous compound or solvated compound, or a pharmaceutical composition for use in therapy.

[0015] A further aspect of the present invention provides a compound as defined herein or an agent-acceptable salt, aqueous compound or solvated compound thereof, or a pharmaceutical composition as defined herein, for use in the treatment of a proliferative condition.

[0016] A further aspect of the present invention provides a compound as defined herein or an agent-acceptable salt thereof, a aqueous compound or solvated compound, or a pharmaceutical composition for use in the treatment of cancer.

[0017] A further aspect of the present invention provides compounds as defined herein or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof used in inhibiting LMO2 activity.

[0018] A further aspect of the present invention provides compounds as defined herein or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof, which are used in the treatment of diseases or disorders related to LMO2 activity.

[0019] A further aspect of the present invention provides the use of compounds defined herein or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof in the manufacture of drugs for the treatment of proliferative conditions.

[0020] Appropriately, proliferative disorders are cancers, specifically human cancers. Specific examples of appropriate cancers include, but are not limited to, any cancers involving LMO2 activity, such as hematological cancers, e.g., lymphomas (diffuse large B-cell lymphoma (DLBCL), B-cell acute lymphoblastic lymphoma (B-ALL), follicular lymphoma (FL), Burkitt lymphoma (BL), and angioimmunoblastic T-cell lymphoma (AITL)), leukemias (acute lymphoblastic leukemia (ALL) that induces T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML)), and multiple myeloma.

[0021] A further aspect of the present invention provides the use of compounds defined herein or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof in the manufacture of drugs for the treatment of cancer.

[0022] A further aspect of the present invention provides the use of a compound as defined herein or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof in the production of a drug for inhibiting LMO2 activity.

[0023] A further aspect of the present invention provides for the use of compounds defined herein or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof in the manufacture of drugs for the treatment of diseases or disorders related to LMO2 activity.

[0024] A further aspect of the present invention provides a process for producing a compound as defined herein or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof.

[0025] Further aspects of the present invention provide compounds or pharmaceutically acceptable salts, aqueous compounds or solvated compounds thereof that can be obtained, obtained, or directly obtained by processes for producing compounds as defined herein.

[0026] A further aspect of the present invention provides a novel intermediate, as defined herein, suitable for use in any one of the synthesis methods described herein.

[0027] A feature comprising an optional, appropriate, and preferred feature relating to one aspect of the present invention may also be a feature comprising an optional, appropriate, and preferred feature relating to any other aspect of the present invention. [Modes for carrying out the invention]

[0028] [Definition] Unless otherwise specified, the following terms used in this specification and in the claims shall have the meanings set forth below.

[0029] It should be understood that the reference to “treating” or “treatment” includes the prevention and reduction of established symptoms of a condition. Therefore, “treating” or “treatment” of a condition, disease, or medical condition includes: (1) preventing or delaying the onset of clinical symptoms of a condition, disease, or medical condition in a person who has or is predisposed to the condition, disease, or medical condition but has not yet experienced or delayed any clinical or subclinical symptoms of the condition, disease, or medical condition; (2) inhibiting the condition, disease, or medical condition, i.e., preventing, reducing, or delaying the onset of the disease or relapse (in the case of maintenance treatment), or at least one clinical or subclinical symptom thereof; or (3) reducing or attenuating the disease, i.e., causing the regression of at least one clinical or subclinical symptom of the condition, disease, or medical condition.

[0030] The "therapeutic dose" refers to the amount of a compound that, when administered to a mammal to treat a disease, is sufficient to act as a therapeutic agent for that disease. The therapeutic dose may vary depending on the compound, the disease and its severity, and the age, weight, etc., of the mammal being treated.

[0031] In this specification, the term “alkyl” includes both linear and branched alkyl groups. References to individual alkyl groups, such as “propyl,” refer only to the linear version, and references to individual branched alkyl groups, such as “isopropyl,” refer only to the branched version. For example, “(1-6C)alkyl” includes (1-4C)alkyl, (1-3C)alkyl, propyl, isopropyl, and t-butyl.

[0032] The terms "(m~nC)" or "(m~nC) group," used alone or as a prefix, refer to a group having m to n carbon atoms.

[0033] The "alkylene" group is an alkyl group located between two other chemical groups, playing a role in linking them together. Therefore, "(1-6C)alkylene" refers to a linear saturated divalent hydrocarbon radical with 1 to 6 carbon atoms, or a branched saturated divalent hydrocarbon radical with 3 to 6 carbon atoms, such as methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), 2-methylpropylene (-CH2CH(CH3)CH2-), pentylene (-CH2CH2CH2CH2CH2-), etc.

[0034] The term "alkenyl" refers to a linear or branched alkyl group containing two or more carbon atoms, with at least one intercarbon double bond present within its group. Examples of alkenyl groups include ethenyl, propenyl, and buto-2,3-enyl, encompassing all possible geometric (E / Z) isomers.

[0035] The term "alkynyl" refers to a linear or branched alkyl group containing two or more carbon atoms, with at least one triple bond between carbon atoms present within the group. Examples of alkynyl groups include cetirenyl and propynyl.

[0036] "(3-10C) cycloalkyl" means a hydrocarbon ring containing 3 to 10 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and bicyclo[2.2.1]heptyl.

[0037] The term "alkoxy" refers to O-linked linear and branched alkyl groups. Examples of alkoxy groups include methoxy, ethoxy, and t-butoxy.

[0038] The term "haloalkyl" is used herein to mean an alkyl group in which one or more hydrogen atoms are substituted by halogen (e.g., fluorine) atoms. Examples of haloalkyls include -CH2F, -CHF2, and -CF3.

[0039] The terms "halo" or "halogeno" mean fluoro, chloro, bromo and iodine, more appropriately fluoro, chloro and bromo, and more appropriately fluoro and chloro.

[0040] The terms "carbocykrill," "carbocyclic," or "carbocyclic" refer to non-aromatic saturated or partially saturated monocyclic, condensed, cross-linked, or spiro-dicyclic carbon-containing ring structures. Monocyclic carbocyclic rings contain 3 to 12 (appropriately 3 to 7) ring atoms. Bicyclic carbocyclic rings contain 6 to 17 (appropriately 7 to 12) member atoms in their ring. Bicyclic carbocyclic rings can be condensed, spiro, or cross-linked ring structures. Examples of carbocyclic groups include cyclopropyl, cyclobutyl, cyclohexyl, cyclohexenyl, and spiro[3.3]heptanyl.

[0041] The terms "heterocyclyl," "heterocyclic," or "heterocyclic" refer to non-aromatic saturated or partially saturated monocyclic, condensed, bridged, or spiro-dicyclic heterocyclic ring structures. Monocyclic heterocyclic rings contain approximately 3 to 12 (preferably 3 to 7) ring atoms, with 1 to 5 (preferably 1, 2, or 3) heteroatoms in the ring selected from nitrogen, oxygen, or sulfur. Bicyclic heterocyclic rings contain 7 to 17 (preferably 7 to 12) member atoms in the ring. Bicyclic heterocyclic rings can be condensed spiro or bridged ring structures. Examples of heterocyclic groups include cyclic ethers, oxylanyls, oxetanyls, tetrahydrofuranyls, dioxanyls, and substituted cyclic ethers. Examples of nitrogen-containing heterocyclic rings include azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydrotriazinyl, and tetrahydropyrazolyl. Common sulfur-containing heterocycles include tetrahydrothienyl, dihydro-1,3-dithiol, tetrahydro-2H-thiopyran, and hexahydrothiepine. Other heterocycles include dihydrooxathiolyl, tetrahydrooxazolyl, tetrahydro-oxadiazolyl, tetrahydrodioxazolyl, tetrahydrooxazolyl, hexahydrotriazinyl, tetrahydrooxazinyl, morpholinyl, thiomorpholinyl, tetrahydropyrimidinyl, dioxolinyl, octahydrobenzofuranyl, octahydrobenzimidazolyl, and octahydrobenzothiazolyl. Regarding sulfur-containing heterocycles, sulfur oxide heterocycles containing SO or SO2 groups are also included. Examples include the sulfoxide and sulfone forms of tetrahydrothienyl and thiomorpholinyl, such as tetrahydrothienyl 1,1-dioxide and thiomorpholinyl 1,1-dioxide. The heterocycle may contain one or two oxo (=O) or thioxo (=S) substituents. Suitable values ​​for the heterocyclyl group having one or two oxo (=O) or thioxo (=S) substituents are, for example, 2-oxopyrrolidinyl, 2-thioxopyrrolidinyl, 2-oxoimidazolidinyl, 2-thioxoimidazolidinyl, 2-oxopiperidinyl, 2,5-dioxopyrrolidinyl, 2,5-dioxoimidazolidinyl, or 2,6-dioxopiperidinyl.Certain heterocyclyl groups are saturated monocyclic 3- to 7-membered heterocyclyls containing 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, or sulfur, such as azetidinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, morpholinyl, tetrahydrothienyl, tetrahydrothienyl 1,1-dioxide, thiomorpholinyl, thiomorpholinyl 1,1-dioxide, piperidinyl, homopiperidinyl, piperazinyl, or homopiperazinyl. As can be understood by those skilled in the art, any heterocycle can be linked to another group via a suitable atom, such as a carbon or nitrogen atom. However, references to piperidino or morpholino herein mean piperidin-1-yl or morpholin-4-yl rings linked via ring nitrogen.

[0042] A "bridged ring structure" refers to a ring structure in which two rings share more than two atoms; see, for example, Jerry March, Advanced Organic Chemistry, by [name unclear], 4th Edition, Wiley Interscience, pages 131-133, 1992. Examples of bridged heterocyclyl ring structures include azabicyclo[2.2.1]heptane, 2-oxa-5-azabicyclo[2.2.1]heptane, azabicyclo[2.2.2]octane, azabicyclo[3.2.1]octane, and quinuclidine.

[0043] The inventors define a "spiro-bicyclic ring structure" as a structure in which two ring structures share one common spirocarbon atom, that is, a heterocyclic ring is linked to a further carbocyclic or heterocyclic ring via one common spirocarbon atom. Examples of spiro-ring structures include 6-azaspiro[3.4]octane, 2-oxa-6-azaspiro[3.4]octane, 2-azaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.3]heptane, 7-oxa-2-azaspiro[3.5]nonane, 6-oxa-2-azaspiro[3.4]octane, 2-oxa-7-azaspiro[3.5]nonane, and 2-oxa-6-azaspiro[3.5]nonane.

[0044] The terms "heteroaryl" or "heteroaromatic" refer to aromatic monocyclic, bicyclic, or polycyclic rings incorporating one or more heteroatoms (e.g., particularly one, two, or three) selected from nitrogen, oxygen, or sulfur. The term heteroaryl includes both monovalent and divalent species. Examples of heteroaryl groups are monocyclic and bicyclic groups containing 5 to 12 ring members, more typically 5 to 10. Heteroaryl groups can be, for example, 5- or 6-membered monocyclic rings or 9- or 10-membered bicyclic rings, such as bicyclic structures formed from fused 5- and 6-membered rings or two fused 6-membered rings. Each ring may contain about four heteroatoms, typically selected from nitrogen, sulfur, and oxygen. Generally, heteroaryl rings will contain up to three heteroatoms, more typically up to two, for example, one heteroatom. In one embodiment, the heteroaryl ring contains at least one ring nitrogen atom. Nitrogen atoms in a heteroaryl ring can be basic, as in the case of imidazole or pyridine, or they can be essentially non-basic, as in the case of indole or pyrrole nitrogen. In general, the number of basic nitrogen atoms present in a heteroaryl group containing any amino group substituent in the ring will be less than five.

[0045] Examples of heteroaryls include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridadinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazeninyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, prinyl, benzoflazanil, quinolyl, i Examples include soquinoryl, quinazolinil, quinoxalinil, sinnolinil, pteridinil, naphthilidinil, carbazolyl, phenadinil, benzisoquinolinil, pyridopyrazine, thieno[2,3b]furanil, 2H-furo[3,2b]pyranil, 5H-pyrido[2,3d]ooxazinil, 1H-pyrazolo[4,3d]oxazolyl, 4H-imidazo[4,5d]thiazolyl, pyrazino[2,3d]pyridazinil, imidazo[2,1b]thiazolyl, and imidazo[1,2b][1,2,4]triazinil. "Heteroaryl" also includes partially aromatic bicyclic or polycyclic ring structures in which at least one ring is aromatic and one or more of the other rings are non-aromatic, saturated, or partially saturated, provided that at least one ring contains one or more heteroatoms selected from nitrogen, oxygen, or sulfur. Examples of partially aromatic heteroaryl groups include, for example, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 2-oxo-1,2,3,4-tetrahydroquinolinyl, dihydrobenzthienyl, dihydrobenzfuranyl, 2,3-dihydro-benzo[1,4]dioxynyl, benzo[1,3]dioxolyl, 2,2-dioxo-1,3-dihydro-2-benzothienyl, 4,5,6,7-tetrahydrobenzofuranyl, indlinyl, 1,2,3,4-tetrahydro1,8-naphthilidinyl, 1,2,3,4-tetrahydropyrido[2,3b]pyradinyl, and 3,4-dihydro2Hpyrido[3,2b][1,4]oxazinyl.

[0046] Examples of five-membered heteroaryl groups include, but are not limited to, pyrrolyl, furanil, thienyl, imidazolyl, furazanil, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl, and tetrazolyl groups.

[0047] Examples of six-membered heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyridadinyl, pyrimidinyl, and triazinyl.

[0048] Bicyclic heteroaryl groups are, for example: A benzene ring condensed into a five-membered or six-membered ring containing one, two, or three ring heteroatoms; A pyridine ring condensed into a five-membered or six-membered ring containing one, two, or three ring heteroatoms; A pyrimidine ring condensed into a five-membered or six-membered ring containing one or two ring heteroatoms; A pyrrole ring condensed into a five-membered or six-membered ring containing one, two, or three ring heteroatoms; A pyrazole ring condensed into a five- or six-membered ring containing one or two ring heteroatoms; A pyrazine ring condensed into a five-membered or six-membered ring containing one or two ring heteroatoms; An imidazole ring condensed into a five-membered or six-membered ring containing one or two ring heteroatoms; An oxazole ring condensed into a five-membered or six-membered ring containing one or two ring heteroatoms; An isoxazole ring condensed into a five-membered or six-membered ring containing one or two ring heteroatoms; A thiazole ring condensed into a five-membered or six-membered ring containing one or two ring heteroatoms; An isothiazole ring condensed into a five-membered or six-membered ring containing one or two ring heteroatoms; A thiophene ring condensed into a five-membered or six-membered ring containing one, two, or three ring heteroatoms; A furan ring fused to a five-membered or six-membered ring containing one, two, or three ring heteroatoms; A cyclohexyl ring fused to a 5-membered or 6-membered aromatic heterocycle containing 1, 2, or 3 ring heteroatoms; and A cyclopentyl ring fused to a five-membered or six-membered aromatic heterocycle containing one, two, or three ring heteroatoms; It can be a base selected from among them.

[0049] Specific examples of bicyclic heteroaryl groups containing a six-membered ring condensed onto a five-membered ring include, but are not limited to, benzfuranil, benzthiophenyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzthiazolyl, benzisothiazolyl, isobenzofuranil, indolyl, isoindolyl, indolidinyl, indolinyl, isoindolinyl, prinyl (e.g., adeninyl, guaninyl), indazolyl, benzodioxolyl, and pyrazolopyridinyl groups.

[0050] Specific examples of bicyclic heteroaryl groups containing two fused six-membered rings include, but are not limited to, quinolinyl, isoquinolinyl, chromanyl, thiochromanyl, chromenyl, isochromenyl, chromanyl, isochromanyl, benzodioxanyl, quinolidinyl, benzoxazinyl, benzodiadinyl, pyridopyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthilidinyl, and pteridinyl groups.

[0051] The term "aryl" refers to a cyclic or polycyclic aromatic ring having 5 to 12 carbon atoms. The term aryl includes both monovalent and divalent forms. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, and naphthyl. In certain embodiments, the aryl is phenyl.

[0052] In this specification, several compound terms are also used to describe groups that include multiple functionalities. Such terms will be understood by those skilled in the art. For example, (3-6C)cycloalkyl(m-nC)alkyl includes (m-nC)alkyls substituted with (3-6C)cycloalkyls.

[0053] The term "optionally substituted" means any group, structure, or molecule that is substituted, and any group, structure, or molecule that is not substituted. "R" 1 The expression "one / any of the CH, CH2, CH3 groups or heteroatoms (i.e., NH) in the group is optionally substituted" properly means that 1 one (any) of the hydrogen radicals of the group is substituted by a group according to the relevant regulations.

[0054] When an optional substituent is selected from "one or more" groups, it should be understood that this definition includes all substituents selected from one of the specified groups or substituents selected from two or more of the specified groups.

[0055] The phrase "the compounds of the present invention" generally and specifically means the compounds disclosed herein.

[0056] <Compounds of the present invention> In one aspect, the present invention relates to the following structural formula (I):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0057] The specific compounds of the present invention include, for example, compounds of formula (I), or pharmaceutically acceptable salts, hydrates and / or solvates thereof. Unless otherwise specified, each of R1, X1, X2, X3, Q, R2, R3 and R4 and any related substituents has any of the meanings defined above or defined in any of the following paragraphs (1) to (22): (1) R1 is: (ii) (1-4C) alkyl optionally substituted by one or more R a ; (ii) a group of the formula:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0058] Appropriately, R1 is defined as in any one of the above paragraphs (4) to (7). Most appropriately, R1 is defined in paragraph (6) or paragraph (7).

[0059] Appropriately, X1 is defined as in any one of the above paragraphs (8) to (10). Most appropriately, X1 is defined as in paragraph (10) above.

[0060] Appropriately, X2 is defined as in any one of the above paragraphs (8) to (10). Most appropriately, X2 is defined as in paragraph (10) above.

[0061] Appropriately, X3 is defined as in any one of the above paragraphs (8) to (10). Most appropriately, X3 is defined as in paragraph (10) above.

[0062] Appropriately, X1, X2, and X3 are defined as in any one of the above paragraphs (8) to (10). Most appropriately, X1, X2, and X3 are defined as in paragraph (10) above.

[0063] Appropriately, Q is defined as in any one of the above paragraphs (11) to (14). Most appropriately, Q is defined as in paragraph (14) above.

[0064] Appropriately, R2 is defined in paragraphs (15) and (16) above. Most appropriately, R2 is defined in paragraph (16) above.

[0065] Appropriately, R3 is defined in paragraphs (15) and (16) above. Most appropriately, R3 is defined in paragraph (16) above.

[0066] Appropriately, R2 and R3 are defined as in paragraphs (15) and (16) above. Most appropriately, R2 and R3 are defined in paragraph (16) above.

[0067] Appropriately, R4 is defined as in any one of the above paragraphs (19) to (22). Most appropriately, R4 is defined as in paragraph (21) or paragraph (22) above.

[0068] In a specific group of compounds of the present invention, R2 and R3 are hydrogen, and Q is [ka] And in the formula, [ka] This indicates the connection point. The compound, or its pharmaceutically acceptable salts, aqueous compounds, and / or solvated compounds, are shown in the following structural formula Ia (sub-definition of formula (I)): [ka] It has, In the above formula, R1, X1, X2, X3, and R4 are as defined above.

[0069] In one embodiment of the compound of formula Ia: R1 is defined in any one of the above paragraphs (1) to (7); X1, X2, and X3 are defined in any one of the above paragraphs (8) to (10); and R4 is defined in any one of the above paragraphs (17) to (22).

[0070] In another embodiment of the compound of formula Ia: R1 is defined as in paragraph (4) above; X1, X2, and X3 are as defined in paragraph (8) above; and R4 is defined in paragraph (18) above.

[0071] In another embodiment of the compound of formula Ia: R1 is defined as in paragraph (5) above; X1, X2, and X3 are as defined in paragraph (9) above; and R4 is defined as described in paragraph (19) above.

[0072] In another embodiment of the compound of formula Ia: R1 is defined as in paragraph (6) or paragraph (7) above; X1, X2, and X3 are as defined in paragraph (10) above; and R4 is defined in paragraph (21) or paragraph (22) above.

[0073] In another embodiment of the compound of formula Ia: R1 is defined as in paragraph (6) above; X1, X2, and X3 are as defined in paragraph (10) above; and R4 is defined as described in paragraph (21) above.

[0074] In another embodiment of the compound of formula Ia: R1 is defined as in paragraph (7) above; X1, X2, and X3 are as defined in paragraph (10) above; and R4 is defined as described in paragraph (22) above.

[0075] In a specific group of compounds of the present invention, the compound, or its pharmaceutically acceptable salt, aqueous compound, and / or solvated compound is shown in the following structural formula Ib (sub-definition of formula (I)): [ka] It has, In the formula, R1, X1, X2, X3, and R4 are as defined above.

[0076] In one embodiment of the compound of formula Ib: R1 is defined in any one of the above paragraphs (1) to (7); X1, X2, and X3 are defined in any one of the above paragraphs (8) to (10); and R4 is defined in any one of the above paragraphs (17) to (22).

[0077] In another embodiment of the compound of formula Ib: R1 is defined as in paragraph (4) above; X1, X2, and X3 are as defined in paragraph (8) above; and R4 is defined in paragraph (18) above.

[0078] In another embodiment of the compound of formula Ib: R1 is defined as in paragraph (5) above; X1, X2, and X3 are as defined in paragraph (9) above; and R4 is defined as described in paragraph (19) above.

[0079] In another embodiment of the compound of formula Ib: R1 is defined as in paragraph (6) or paragraph (7) above; X1, X2, and X3 are as defined in paragraph (10) above; and R4 is defined in paragraph (21) or paragraph (22) above.

[0080] In another embodiment of the compound of formula Ib: R1 is defined as in paragraph (6) above; X1, X2, and X3 are as defined in paragraph (10) above; and R4 is defined as described in paragraph (21) above.

[0081] In another embodiment of the compound of formula Ib: R1 is defined as in paragraph (7) above; X1, X2, and X3 are as defined in paragraph (10) above; and R4 is defined as described in paragraph (22) above.

[0082] In a specific group of compounds of the present invention, the compound, or its pharmaceutically acceptable salt, aqueous compound, and / or solvated compound, is defined as having the following structural formula Ic (sub-definition of formula (I)): [ka] It has, In the above formula, R1, X1, X2, X3, and R4 are as defined above.

[0083] In one embodiment of the compound of formula Ic: R1 is defined in any one of the above paragraphs (1) to (7); Q is defined as in any one of the above paragraphs (11) to (14); R2 and R3 are as defined in paragraphs (15) and (16) above; and R4 is defined in any one of the above paragraphs (17) to (22).

[0084] In another embodiment of the compound of formula Ic: R1 is defined as in paragraph (4); Q is defined as in paragraph (12); R2 and R3 are as defined in paragraph (15) above; and R4 is defined in paragraph (18) above.

[0085] In another embodiment of the compound of formula Ic: R1 is defined in paragraph (5); Q is defined as in paragraph (12); R2 and R3 are as defined in paragraph (15) above; and R4 is defined as described in paragraph (19) above.

[0086] In another embodiment of the compound of formula Ic: R1 is as defined in paragraph (6) or paragraph (7); Q is defined as in paragraph (14); R2 and R3 are as defined in paragraph (16) above; and R4 is defined in paragraph (21) or paragraph (22) above.

[0087] In another embodiment of the compound of formula Ic: R1 is defined in paragraph (6); Q is defined as in paragraph (14); R2 and R3 are as defined in paragraph (16) above; and R4 is defined as described in paragraph (21) above.

[0088] In another embodiment of the compound of formula Ic: R1 is defined in paragraph (7); Q is defined as in paragraph (14); R2 and R3 are as defined in paragraph (16) above; and R4 is defined as described in paragraph (22) above.

[0089] In a specific group of compounds of the present invention, the compound, or its pharmaceutically acceptable salt, aqueous compound, and / or solvated compound, is defined as the following structural formula Id (sub-definition of formula (I)): [ka] It has, In the above formula, R1, Q, R2, R3, and R4 are as defined above.

[0090] In one embodiment of the compound of formula Id: R1 is defined in any one of paragraphs (1) to (7); Q is defined as in any one of paragraphs (11) to (14); R2 and R3 are as defined in paragraphs (15) and (16) above; and R4 is defined in any one of the above paragraphs (17) to (22).

[0091] In another embodiment of the compound of formula Id: R1 is defined as in paragraph (4); Q is defined as in paragraph (12); R2 and R3 are as defined in paragraph (15) above; and R4 is defined in paragraph (18) above.

[0092] In another embodiment of the compound of formula Id: R1 is defined in paragraph (5); Q is defined as in paragraph (12); R2 and R3 are as defined in paragraph (15) above; and R4 is defined as described in paragraph (19) above.

[0093] In another embodiment of the compound of formula Id: R1 is as defined in paragraph (6) or paragraph (7); Q is defined as in paragraph (14); R2 and R3 are as defined in paragraph (16) above; and R4 is defined in paragraph (21) or paragraph (22) above.

[0094] In another embodiment of the compound of formula Id: R1 is defined in paragraph (6); Q is defined as in paragraph (14); R2 and R3 are as defined in paragraph (16) above; and R4 is defined as described in paragraph (21) above.

[0095] In another embodiment of the compound of formula Id: R1 is defined in paragraph (7); Q is defined as in paragraph (14); R2 and R3 are as defined in paragraph (16) above; and R4 is defined as described in paragraph (22) above.

[0096] In a particular group of compounds of the present invention, X1, X2, and X3 are as defined in paragraph (10) above, Q is as defined in paragraph (14) above, and R2 and R3 are as defined in paragraph (15) above: R1 is defined in any one of the above paragraphs (1) to (7); R4 is defined in any one of the above paragraphs (17) to (22).

[0097] In a further group of compounds of the present invention, X1, X2, and X3 are as defined in paragraph (10) above, Q is as defined in paragraph (14) above, and R2 and R3 are as defined in paragraph (15) above: R1 is defined as in paragraph (5) above; R4 is defined in paragraph (18) above.

[0098] In a further group of compounds of the present invention, X1, X2, and X3 are as defined in paragraph (10) above, Q is as defined in paragraph (14) above, and R2 and R3 are as defined in paragraph (15) above: R1 is defined as in paragraph (5) above; R4 is defined as described in paragraph (19) above.

[0099] In a further group of compounds of the present invention, X1, X2, and X3 are as defined in paragraph (10) above, Q is as defined in paragraph (14) above, and R2 and R3 are as defined in paragraph (15) above: R1 is defined as in paragraph (6) above; R4 is defined as described in paragraph (20) above.

[0100] In a further group of compounds of the present invention, X1, X2, and X3 are as defined in paragraph (10) above, Q is as defined in paragraph (14) above, and R2 and R3 are as defined in paragraph (15) above: R1 is defined as in paragraph (6) above; R4 is defined as described in paragraph (21) above.

[0101] In a further group of compounds of the present invention, X1, X2, and X3 are as defined in paragraph (10) above, Q is as defined in paragraph (14) above, and R2 and R3 are as defined in paragraph (15) above: R1 is defined as in paragraph (7) above; R4 is defined as described in paragraph (22) above.

[0102] The specific compounds of the present invention include any of the compounds exemplified herein, or their pharmaceutically acceptable salts or solvated compounds, and in particular the following: 2-(3-benzyl-2-oxoimidazolidine-1-yl)-N-(4-phenoxyphenyl)oxazole-4-carboxamide (Abd-L6); N-(4-(1H-pyrrole-1-yl)phenyl)-2-(3-(3-chlorobenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxamide (Abd-L7); 2-(3-(4-chlorobenzyl)-2-oxoimidazolidine-1-yl)-N-(3,4-dimethoxyphenyl)oxazole-5-carboxamide (Abd-L8); N-(4-(benzyloxy)phenyl)-2-(3-(3-methoxybenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxamide (Abd-L9); N-(4-(1H-pyrrole-1-yl)phenyl)-2-(3-(3-cyanobenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxamide(Abd-L10); 2-(3-(2-chlorobenzyl)-2-oxoimidazolidine-1-yl)-N-(4-phenoxyphenyl)oxazole-4-carboxamide(Abd-L12); 2-(3-benzyl-2-oxoimidazolidine-1-yl)-N-(4-phenoxybenzyl)oxazole-4-carboxamide (Abd-L13); 2-(3-(2-chlorobenzyl)-2-oxoimidazolidine-1-yl)-N-(4-phenoxybenzyl)oxazole-4-carboxamide (Abd-L14); N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(3-methoxybenzyl)piperazine-1-yl)oxazole-4-carboxamide (Abd-L15); N-(4-(benzyloxy)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)oxazole-4-carboxamide (Abd-L16); N-(4-(benzyloxy)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)thiazole-4-carboxamide (Abd-L17); N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(3-methoxybenzyl)piperazine-1-yl)thiazole-4-carboxamide (Abd-L18); N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(4-methoxybenzyl)piperazine-1-yl)oxazole-4-carboxamide (Abd-L19); N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(2-methoxybenzyl)piperazine-1-yl)thiazole-4-carboxamide (Abd-L20); 2-(4-(3-methoxybenzyl)piperazin-1-yl)-N-(4-(trifluoromethoxy)phenyl)thiazole-4-carboxamide (Abd-L21); 2-(4-(3-methoxybenzyl)piperazine-1-yl)-N-(6-methoxypyridine-3-yl)thiazole-4-carboxamide (Abd-L22); and 2-(4-(3-methoxybenzyl)piperazine-1-yl)-N-(2-methoxypyrimidine-5-yl)thiazole-4-carboxamide (Abd-L23); Includes any of the following.

[0103] The various functional groups and substituents constituting the compounds of formula (I) or subformulas Ia to Id are generally selected so that the molecular weight of the compound of formula (I) does not exceed 1000. More typically, the molecular weight of the compound is less than 900, for example less than 800, or less than 750, or less than 700, or less than 650. More preferably, the molecular weight is less than 600, for example 550 or less.

[0104] Suitable pharmacopoeciable salts of the compounds of the present invention include, for example, acid addition salts of the compounds of the present invention that are sufficiently basic, such as acid addition salts with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, trifluoroacetic acid, formic acid, methanesulfonic citrate, or maleic acid. Furthermore, suitable pharmacopoeciable salts of the compounds of the present invention that are sufficiently acidic include alkali metal salts, such as sodium or potassium salts, alkaline earth metal salts, such as calcium or magnesium salts, ammonium salts, or salts with organic bases that give pharmacopoeciable cations, such as methylamine, dimethylamine, trimethylamine, piperidine, morpholine, or tris-(2-hydroxyethyl)amine.

[0105] Compounds that have the same molecular formula but differ in the nature or order of their atomic bonding or the spatial arrangement of their atoms are called "isomers." Stereoisomers that are not mirror images of each other are called "diastereoisomers," and stereoisomers that are mirror images of each other but cannot be superimposed are called "enantiomers." If a compound has a chiral center, for example, it may be bonded to four different groups, and a pair of enantiomers are possible. Enantiomers are characterized by the absolute configuration of their chiral center. This is described by the Cahn and Prelog R and S order rules, or by the method of the molecule rotating its plane of polarization, and is designated as dextrorotatory or levorotatory (i.e., as (+) or (-) isomers, respectively). Chiral compounds can exist as individual enantiomers or as mixtures thereof. A mixture containing enantiomers in equal proportions is called a "racemic mixture."

[0106] The compounds of the present invention may have one or more chiral centers; therefore, such compounds may be produced as individual (R) or (S) stereoisomers, or as mixtures thereof. Unless otherwise specified, the descriptions and nomenclature of specific compounds in this specification and claims are intended to encompass both individual enantiomers and their mixtures, racemic forms, or other forms. For example, methods for determining stereochemistry and separating stereoisomers by synthesis from optically active starting materials or by resolving racemic forms are well known in the art (see the discussion in Chapter 4 of Advanced Organic Chemistry, 4th edition J. March, John Wiley and Sons, New York, 2001). Some of the compounds of the present invention may have geometric isomer centers (E and Z isomers).

[0107] It should be understood that this invention encompasses all optical diastereoisomers and geometric isomers, as well as mixtures thereof, that possess antiproliferative activity.

[0108] The present invention also includes compounds of the present invention as defined herein that involve one or more isotopic substitutions. For example, H may take any isotopic form such as 1H, 2H(D), and 3H(T); C may take any isotopic form such as 12C, 13C, and 14C; and O may take any isotopic form such as 16O and 18O, etc.

[0109] It should be understood that certain compounds of formula (I) or subformulas Ia to Id may exist in both solvated and non-solvated forms, such as hydrated forms. It should be understood that the present invention encompasses all such solvated forms that possess antiproliferative activity.

[0110] It should be understood that certain compounds of formula (I) or subformulas Ia to Id may exhibit polymorphism, and that the present invention encompasses all such forms that possess antiproliferative activity.

[0111] Compounds of formula (I) or subformulas Ia-Id may exist in many different tautomers, and a reference to a compound of formula (I) or subformulas Ia-Id encompasses all such forms. To avoid ambiguity, if a compound may exist in one of several tautomers and only one is specifically described or shown, then all others are nevertheless encompassed by formula (I) or subformulas Ia-Id. Examples of tautomers include, for example, the following tautomer pairs: keto / enol (illustrated below), imine / enamine, amide / iminoalcohol, amidine / amidine, nitroso / oxime, thioketone / enthiol, and nitro / acy-nitro, as well as the keto, enol, and enolate forms. [ka]

[0112] Compounds of formula (I) or subformulas Ia-Id containing amine functional groups may also form N-oxides. References to compounds of formula (I) or subformulas Ia-Id containing amine functional groups include N-oxides. If a compound contains several amine functional groups, one or more nitrogen atoms may be oxidized to form an N-oxide. Specific examples of N-oxides are tertiary amines, or N-oxides of nitrogen atoms in nitrogen-containing heterocycles. N-oxides can be formed by treatment with the corresponding amine with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., a peroxycarboxylic acid); see, for example, Jerry March, Advanced Organic Chemistry, 4th Edition, Wiley Interscience, pages. Further details show that N-oxides can be produced by the procedure of LWDeady (Syn.Comm.1977,7,509-514), in which amine compounds are reacted in an inert solvent such as m-chloroperoxybenzoic acid (mCPBA), e.g., dichloromethane.

[0113] Compounds of formula (I) or subformulas Ia-Id may be administered in the form of a prodrug, which is broken down in the body of a human or animal to release the compound of the present invention. The physical and / or pharmacokinetic properties of the present invention may be altered using the prodrug. A prodrug can be formed if the compound of the present invention contains a suitable group or substituent to which a property-modifying group can be attached. Examples of prodrugs include in vivo cleavable ester derivatives that can be formed at the carboxyl or hydroxyl group of the compound of formula (I) or subformulas Ia-Id, and in vivo cleavable amide derivatives that can be formed at the carboxyl or amino group of the compound of formula (I) or subformulas Ia-Id.

[0114] Accordingly, the present invention includes compounds of formula (I) or subformulas Ia-Id as defined above, when made available by organic synthesis and when made available in the body of a human or animal by cleavage of its prodrug. Accordingly, the present invention includes compounds of formula (I) or subformulas Ia-Id produced by organic synthesis means, and such compounds produced in the body of a human or animal by the metabolism of a precursor compound which is a compound of formula (I) or subformulas Ia-Id may also be synthetically produced compounds or metabolically produced compounds.

[0115] A prodrug that is acceptable as a suitable agent is one that is deemed suitable for administration to the human or animal body without undesirable pharmacological activity and without excessive toxicity, based on reasonable medical judgment.

[0116] Various forms of prodrugs are described in the following document, for example: a)Methods in Enzymology,Vol.42,p.309-396,edited by K.Widder,et al.(Academic Press,1985); b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985); c)A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H.Bundgaard,Chapter 5 “Design and Application of Pro-drugs”, by H.Bundgaard p.113-191(1991); d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); e) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); f) N. Kakeya, et al.,Chem.Pharm.Bull.32,692(1984); g) T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”, ACSSymposium Series, Volume 14; and h) E. Roche (editor), “Bioreversible Carriers in Drug Design”, Pergamon Press, 1987 It is described there.

[0117] Suitable pharmacovigilant prodrugs of compounds of formula (I) or subformulas Ia-Id that retain a carboxyl group are, for example, esters thereof that are cleavable in vivo. In vivo cleavable esters of compounds of formula I or subformulas Ia-Id that contain a carboxyl group are, for example, pharmacovigilant esters that are cleaved in the body of a human or animal to produce a parent acid. Examples of esters that are acceptable as suitable agents for carboxylation include (1-6C) alkyl esters such as methyl, ethyl, and t-butyl; (1-6C) alkoxymethyl esters such as methoxymethyl; (1-6C) alkanoyloxymethyl esters such as pivaloyloxymethyl and 3-phthalidyl; (3-8C) cycloalkylcarbonyloxy-(1-6C) alkyl esters such as cyclopentylcarbonyloxymethyl and 1-cyclohexylcarbonyloxyethyl; 2-oxo-1,3-dioxolennylmethyl esters such as 5-methyl-2-oxo-1,3-dioxolennylmethyl; and (1-6C) alkoxycarbonyloxy-(1-6C) alkyl esters such as methoxycarbonyloxymethyl and 1-methoxycarbonyloxyethyl.

[0118] Suitable pharmacoagulants that retain a hydroxyl group and are acceptable as pharmaceuticals are, for example, their esters or ethers that are cleavable in vivo. Esters or ethers that contain a hydroxyl group and are cleavable in vivo are, for example, pharmacoagulants that are cleaved in the body of a human or animal to produce a hydroxyl-parent compound. Suitable ester-forming groups for a hydroxyl group include inorganic esters such as phosphate esters (phosphoramidic cyclic esters). Even more suitable ester-forming groups for a hydroxyl group include (1-10C) alkanoyl groups such as acetyl, benzyl, phenylacetyl and substituted benzyl and phenylacetyl groups, and (1-10C) alkoxycarbonyl groups such as ethoxycarbonyl, N,N-(1-6C)2-carbamoyl, 2-dialkylaminoacetyl and 2-carboxyacetyl groups. Examples of ring substituents on phenylacetyl and benzyl groups include aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazine-1-ylmethyl, and 4-(1-4C)alkylpiperazine-1-ylmethyl. Acceptable ether-forming groups suitable for hydroxyl groups include acetoxymethyl and α-acyloxyalkyl groups such as pivaloyloxymethyl.

[0119] Suitable prodrugs acceptable as pharmaceutical agents for compounds of formula (I) or partial formulas Ia-Id that retain a carboxyl group include, for example, amides formed from amines such as ammonia, (1-4C) alkylamines such as methylamine, [(1-4C) alkyl]2 amines such as dimethylamine, N-ethyl N-methylamine or diethylamine, (1-4C) alkoxy(2-4C) alkylamines such as 2-methoxyethylamine, phenyl(1-4C) alkylamines such as benzylamine, and amino acids or esters thereof such as glycine.

[0120] Suitable prodrugs acceptable as agents for compounds of formula (I) or subformulas Ia-Id that retain an amino group are, for example, their amide derivatives that can be cleaved in vivo. Suitable amides acceptable as agents from an amino group are amides formed from (1-10C)alkanoyl groups, such as acetyl, benzyl, phenylacetyl, and substituted benzyl and phenylacetyl groups. Examples of ring substituents on phenylacetyl and benzyl groups include aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazine-1-ylmethyl, and 4-(1-4C)alkyl)piperazine-1-ylmethyl.

[0121] The in vivo effects of compounds of formula (I) or partial formulas Ia-Id may be partially exerted by one or more metabolites formed in the human or animal body after administration of the compound of formula (I) or partial formulas Ia-Id. As described above, the in vivo effects of compounds of formula (I) or partial formulas Ia-Id may also be exerted by the metabolism of a precursor compound (prodrug).

[0122] The present invention may relate to any compound or a particular group of compounds as defined herein, by any optional, preferred, or suitable features, or otherwise with respect to a particular embodiment; however, the present invention may also relate to any compound or a particular group of compounds that specifically exclude the aforementioned optional, preferred, or suitable features or particular embodiment.

[0123] Appropriately, the present invention excludes individual compounds that do not possess the biological activity defined herein.

[0124] <Synthesis> The compounds of the present invention can be produced by any suitable technique known in the art. Specific processes for producing these compounds are further described in the accompanying examples.

[0125] In the description of the synthesis methods described herein, and in the reference synthesis methods used to produce the starting materials, it should be understood that all proposed reaction conditions, including the choice of solvent, reaction atmosphere, reaction temperature, experimental duration, and work-up procedure, can be selected by those skilled in the art.

[0126] Those skilled in organic synthesis will understand that the functional groups present in various parts of a molecule must be compatible with the reagents and reaction conditions used.

[0127] It will be understood that during the synthesis of the compounds of the present invention in the processes defined herein, or during the synthesis of certain starting materials, it is desirable to protect certain substituents to prevent undesirable reactions. It will be understood by chemists in the art when such protection is necessary and how such protecting groups may be placed in place and subsequently removed.

[0128] For examples of protecting groups, see one of the many general texts on the subject, for example, *Protective Groups in Organic Synthesis* by Theodora Green (publisher: John Wiley & Sons). Protecting groups can be removed as appropriate by either a method described in the literature or a simple method known to chemists in the art, such method being chosen to remove the protecting group with minimal interference to other groups in the molecule.

[0129] Therefore, if the reactants contain groups such as amino, carboxy, or hydroxy, it is desirable to protect those groups in part of the reactions described herein.

[0130] As an example, suitable protecting groups for amino or alkylamino groups include, for instance, acyl groups, such as alkanoyl groups like acetyl; alkoxycarbonyl groups, such as methoxycarbonyl, ethoxycarbonyl, or t-butoxycarbonyl groups; arylmethoxycarbonyl groups, such as benzyloxycarbonyl; or aroyl groups, such as benzoyl. The deprotection conditions for the above protecting groups will inevitably differ depending on the choice of protecting group. Therefore, for example, acyl groups such as alkanoyl groups, alkoxycarbonyl groups, or aroyl groups can be removed by hydrolysis with a suitable base, such as alkali metal hydroxides, such as lithium hydroxide or sodium hydroxide. As an alternative method, acyl groups such as t-butoxycarbonyl groups can be removed by treatment with a suitable acid such as hydrochloric acid, sulfuric acid, phosphoric acid, or trifluoroacetic acid, and arylmethoxycarbonyl groups such as benzyloxycarbonyl groups can be removed by hydrogenation with a catalyst such as carbon-supported palladium, or by treatment with a Lewis acid such as boron tris(trifluoroacetate). Suitable alternative protecting groups for primary amino groups are phthaloyl groups, which can be removed by treatment with alkylamines such as dimethylaminopropylamine, or with hydrazine.

[0131] Suitable protecting groups for hydroxyl groups include, for example, acyl groups, such as alkanoyl groups like acetyl, alloyl groups, such as benzoyl, or arylmethyl groups, such as benzyl. The deprotection conditions for the above protecting groups will inevitably vary depending on the choice of protecting group. Therefore, for example, acyl groups such as alkanoyl or alloyl groups can be removed by hydrolysis with a suitable base such as alkali metal hydroxides, such as lithium hydroxide, sodium hydroxide, or ammonia. Alternatively, arylmethyl groups such as benzyl can be removed by hydrogenation with a catalyst such as carbon-supported palladium.

[0132] Suitable protecting groups for carboxyl groups include, for example, esterifying groups such as methyl or ethyl groups that can be removed by hydrolysis with a base such as sodium hydroxide, or t-butyl groups that can be removed by treatment with an acid such as an organic acid such as trifluoroacetic acid, or benzyl groups that can be removed by hydrogenation with a catalyst such as carbon-supported palladium.

[0133] Resins can also be used as protecting groups.

[0134] The methodology used for the synthesis of compounds of formula (I) or subformulas Ia to Id may vary depending on the properties of R1, X1, X2, X3, Q, R2, R3, and R4 and their associated substituents. Suitable processes for their preparation are further described in the accompanying examples.

[0135] When a compound of formula (I) or subformulas Ia to Id is synthesized by any one of the processes defined herein, the process then further includes additional steps: (i) A step of removing any existing protecting group; (ii) A step of converting a compound of formula (I) into another compound of formula (I); (iii) A step of forming a salt, aqueous compound or solvated compound that is acceptable as a pharmaceutical agent; and / or (iv) The process of forming the prodrug; It also includes.

[0136] The example in (ii) above is when a compound of formula (I) is synthesized, and then one or more of the R1, X1, X2, X3, Q, R2, R3, and R4 groups are reacted to change the properties of the group, thereby providing an alternative compound to formula (I).

[0137] The compounds obtained from formula (I) or subformulas Ia to Id can be isolated and purified using techniques well known in the art.

[0138] Compounds of formula (I) can be synthesized by the general synthetic routes shown in the following Examples section, specific examples of which will be described in detail in the Examples.

[0139] <Biological activity> The pharmacological effects of the compounds of the present invention can be measured using the biological assays described in the Examples section of this specification.

[0140] As expected, the pharmacological properties of the compound of formula (I) differ depending on the structural change, but the compound of the present invention was found to be active in the LMO2 in vitro assay described in the Examples section.

[0141] In general, as illustrated by the example compound data in Table 1, the compounds of the present invention are LMO2-iDAb LMO2 as described in the Examples section. dm3 In the BRET assay, except for Abd-L19, the IC50 was 17 μM or less. 50 This indicates that preferred compounds of the present invention, such as Abd-L9, Abd-L10, and Abd-L16, are LMO2-iDAb LMO2. dm3 IC500 is less than 2 μM in the BRET assay. 50 This indicates.

[0142] Furthermore, as illustrated by the example compound data in Table 1, the compounds of the present invention exhibit IC50 or less of 2 μM in the LMO2-LDB1 BRET assay described in the Examples section. 50 This was shown.

[0143] Regarding the examples, the following BRET assay data was generated:

[0144] [Table 1]

[0145] <Pharmaceutical composition> A further aspect of the present invention provides a pharmaceutical composition comprising, in combination with a pharmaceutically acceptable diluent or carrier, the compound of the present invention as defined above, or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof.

[0146] The compositions of the present invention may take the form of dosage forms suitable for oral use (e.g., tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), dosage forms suitable for topical use (e.g., creams, ointments, gels, or aqueous or oily solutions or suspensions), dosage forms suitable for administration by inhalation (e.g., fine powder or liquid aerosol), dosage forms suitable for inhalation (e.g., fine powder), or dosage forms suitable for parenteral administration (e.g., sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular, intraperitoneal, or intramuscular administration, or suppositories for rectal administration).

[0147] The compositions of the present invention are obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Therefore, compositions intended for oral use may contain, for example, one or more colorants, sweeteners, flavorings, and / or preservatives.

[0148] The effective amount of the compound of the present invention used in therapy is sufficient to treat or prevent the proliferative conditions referred to herein, to slow their progression, and / or to reduce the symptoms associated with those conditions.

[0149] The amount of active ingredient combined with one or more excipients to produce a single dose will inevitably vary depending on the individual being treated and the specific route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, 0.5 mg to 0.5 g (more preferably 0.5 to 100 mg, e.g., 1 to 30 mg) of the active ingredient combined with appropriate and convenient amounts of excipients, which may vary from about 5 to about 98% by weight of the total composition.

[0150] The size of therapeutic or prophylactic doses of compounds of formula I will inevitably vary according to well-known medical principles, depending on the nature and severity of the condition, the age and sex of the animal or patient, and the route of administration.

[0151] In the use of the compounds of the present invention for therapeutic or prophylactic purposes, they will generally be administered in divided doses if necessary, receiving a daily dose in the range of, for example, 0.1 to 75 mg / kg body weight. Generally, lower doses will be administered when parenteral routes are used. Thus, for example, in intravenous or intraperitoneal administration, doses in the range of, for example, 0.1 to 30 mg / kg body weight will generally be used. Similarly, in the case of administration by inhalation, doses in the range of, for example, 0.05 to 25 mg / kg body weight will be used. Oral administration may also be suitable, particularly in the form of tablets. Typically, a unit dosage form may contain about 0.5 mg to 0.5 g of the compounds of the present invention.

[0152] <Therapeutic uses and applications> The present invention provides compounds that function as inhibitors of LMO2 activity.

[0153] Accordingly, the present invention provides a method for inhibiting LMO2 activity in vitro or in vivo, the method comprising contacting cells with an effective amount of a compound, or a pharmaceutically acceptable salt thereof, a aqueous compound, or a solvated compound as defined herein.

[0154] The present invention also provides a method for treating a disease or disorder related to LMO2 activity in a patient requiring such treatment, the method comprising administering to the patient a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, a aqueous compound or solvated compound thereof, or a pharmaceutical composition as defined herein.

[0155] The present invention provides a method for inhibiting cell proliferation in vitro or in vivo, the method comprising contacting cells with an effective amount of a compound, or a pharmaceutically acceptable salt thereof, a aqueous compound, or a solvated compound as defined herein.

[0156] The present invention provides a method for treating proliferative disorders in patients requiring such treatment, the method comprising administering to the patient a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, a aqueous compound or solvated compound thereof, or a pharmaceutical composition as defined herein.

[0157] The present invention provides a method for treating cancer in a patient in need of such treatment, the method comprising administering to the patient a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, a aqueous compound or solvated compound, or a pharmaceutical composition as defined herein.

[0158] The present invention provides compounds, or pharmaceutically acceptable salts thereof, aqueous compounds or solvated compounds, or pharmaceutical compositions as defined herein, for use in therapeutics.

[0159] The present invention provides compounds, or pharmaceutically acceptable salts thereof, aqueous compounds or solvated compounds, or pharmaceutical compositions as defined herein, for use in the treatment of proliferative conditions.

[0160] The present invention provides compounds, or pharmaceutically acceptable salts thereof, aqueous compounds or solvated compounds, or pharmaceutical compositions as defined herein, for use in the treatment of cancer.

[0161] The present invention provides compounds, as defined herein, or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof, which are used for inhibiting LMO2 activity.

[0162] The present invention provides compounds, as defined herein, or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof, for use in the treatment of diseases or disorders related to LMO2 activity.

[0163] The present invention provides for the use of compounds, or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof, as defined herein, in the manufacture of drugs for the treatment of proliferative conditions.

[0164] The present invention provides for the use of compounds, or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof, as defined herein, in the manufacture of drugs for the treatment of cancer.

[0165] The present invention provides for the use of compounds, or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof, as defined herein, in the production of drugs for inhibiting LMO2 activity.

[0166] The present invention provides the use of compounds, or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof, as defined herein, in the manufacture of drugs for the treatment of diseases or disorders related to LMO2 activity.

[0167] The terms “proliferative disorders” and “proliferative states” are used herein without distinction and relate to unwanted, excessive, or abnormal cell proliferation, whether in vitro or in vivo, such as neoplastic or hyperplastic proliferation. Examples of proliferative states include, but are not limited to, pre-malignant and malignant cell proliferation, malignant neoplasms and tumors, cancers (breast cancer, non-small cell lung cancer (NSCLC) and squamous cell carcinoma (SCC) (such as SCC of the head and neck, esophagus, lung and ovary), lymphomas (such as diffuse large B-cell lymphoma (DLBCL), B-cell acute lymphoblastic lymphoma (B-ALL), follicular lymphoma (FL), Burkitt lymphoma (BL) and angioimmunoblastic T-cell lymphoma (AITL)), and leukemias (T-cell acute lymphoblastic Examples include acute lymphoblastic leukemia (ALL), including T-ALL, acute myeloid leukemia (AML), and chronic myeloid leukemia (CML), multiple myeloma lymphoma (including acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML)), psoriasis, bone diseases, fibroproliferative disorders (e.g., of connective tissue), and atherosclerosis. However, all types of cells, including those of the lymph, blood, lungs, colon, breast, ovaries, prostate, liver, pancreas, brain, and skin, can be treated.

[0168] The specific proliferative disorders targeted include, for example, lymphomas (diffuse large B-cell lymphoma (DLBCL), B-cell acute lymphoblastic lymphoma (B-ALL), follicular lymphoma (FL), Burkitt lymphoma (BL), and angioimmunoblastic T-cell lymphoma (AITL)), leukemias (acute lymphoblastic leukemia (ALL), including T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML)), and hematological cancers such as multiple myeloma. Diffuse large B-cell lymphoma (DLBCL), B-cell acute lymphoblastic lymphoma (B-ALL), angioimmunoblastic T-cell lymphoma (AITL), T-cell acute lymphoblastic leukemia (T-ALL), and acute myeloid leukemia (AML) are particularly targeted.

[0169] The anticancer effects are not limited to but may occur through one or more mechanisms, including regulation of cell proliferation, inhibition of angiogenesis (formation of new blood vessels), inhibition of diffusion metastasis (spread of tumor from its primary site), inhibition of invasion (spread of tumor cells into adjacent normal structures), or promotion of apoptosis (programmed cell death).

[0170] A compound of formula (I), or a pharmacopoeially acceptable salt thereof, which is an LMO2 inhibitor, has potential therapeutic use in various LMO2-mediated disease conditions.

[0171] Further embodiments of this specification provide compounds of formula (I) as defined above, or pharmaceutically acceptable salts thereof, for use in the treatment of cancer.

[0172] A further feature of this aspect of the Specified is provided for a method of treating cancer in a warm-blooded animal, such as a human, that requires such treatment, the method comprising administering an effective amount of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof.

[0173] Further features of this aspect of the Specified herein are provided for the use of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, in the manufacture of a drug used for the treatment of cancer.

[0174] <Route of administration> The compounds of the present invention or pharmaceutical compositions containing these compounds can be administered to a subject by any convenient route of administration, whether systemic, peripheral, or topical.

[0175] The routes of administration are not limited to, but include oral (e.g., by ingestion); buccal; sublingual; transdermal (e.g., by patch, plaster, etc.); transmucosal (e.g., by patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eye drops); transpulmonary (e.g., through the mouth or nose, e.g., using aerosols, e.g., by inhalation or insulation therapy); transrectal (e.g., by suppositories or enemas); transvaginal (e.g., by pessaries); parenteral administration by injection, e.g., subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intrathecal, intraarticular, intra-articular, subcapsular, intraorbital, intraperitoneal, intratracheal, subepidermal, intraarticular, subarachnoid, and intrasternal administration; and parenteral administration by subcutaneous or intramuscular depot or reservoir implantation, for example.

[0176] <Combination therapy> The growth-inhibiting therapies defined above may be applied as monotherapy, or may include conventional surgery, radiotherapy, or chemotherapy in addition to the compounds of the present invention. Such chemotherapy may fall under the following categories of antitumor agents: (i) Other growth inhibitors / antinomatous drugs and their combinations used in medical oncology, e.g., alkylating agents (e.g., cisplatin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan, temozolomide, and nitrosourea); antimetabolites (e.g., gemcitabine and folate antagonists, fluoropyrimidine derivatives such as 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea); Antitumor antibiotics (e.g., anthracyclines such as adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin C, dactinomycin, and mitramycin); mitotic inhibitors (e.g., vinca alkaloids such as vincristine, vinblastine, vindesine, and vinorelbine, as well as taxoids such as taxol and taxotere, and polokinase inhibitors); and topoisomerase inhibitors (e.g., epipodophyllotoxins such as etoposide and teniposide, amsacrin, topotecan, and camptothecin); (ii) Cell division inhibitors, such as anti-estrogen drugs (e.g., tamoxifen, fulvestrant, toremifene, raloxifene, doroxifene and iodoxifene), anti-androgens (e.g., bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (e.g., goserelin, leupron and buserelin), progestins (e.g., megestrol acetate) and steroid hormones including corticosteroids (e.g., dexamethasone, prednisone and prednisolone), aromatase inhibitors (e.g., anastrozole, letrozole, borazole and exemestane) and 5α-reductase inhibitors such as finasteride; (iii) Anti-soiling agents [e.g., 4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-tetrahydropyran-4-yloxyquinazoline (AZD0530; International Publication No. 01 / 94341), N-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidine-4-ylamino} c-Src kinase family inhibitors such as thiazole-5-carboxamide (dasatinib, BMS-354825; J.Med.Chem., 2004, 47, 6658-6661) and bosutinib (SKI-606), as well as metalloproteinase inhibitors such as marimastat, inhibitors of urokinase-type plasminogen activator receptor function, or antibodies against heparanase. (iv) Inhibitors of growth factor function: For example, such inhibitors include growth factor antibodies and growth factor receptor antibodies (e.g., anti-erbB2 antibody trastuzumab [Herceptin®], anti-EGFR antibody panitumumab, anti-erbB1 antibody cetuximab [Erbitux, C225] and any growth factor or growth factor receptor antibody, as disclosed by Stern et al. (Critical reviews in oncology / haematology, 2005, Vol. 54, pp. 11-29); for example, tyrosine kinase inhibitors, such as inhibitors of the epidermal growth factor family (e.g., N-(3-chloro-4-) EGFR family tyrosine kinase inhibitors such as ruolophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazoline-4-amine (gefitinib, ZD1839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazoline-4-amine (erlotinib, OSI774), and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazoline-4-amine (CI1033), and erbB2 tyrosine inhibitors such as lapatinib. Examples include kinase inhibitors; inhibitors of the hepatocyte growth factor family; inhibitors of the insulin growth factor family; inhibitors of the platelet-derived growth factor family such as imatinib and / or nilotinib (AMN107); serine / threonine kinase inhibitors (e.g., Ras / Raf signaling inhibitors such as farnesyltransferase inhibitors, e.g., sorafenib (BAY43-9006), tipifarnib (R115777), and ronafarnib (SCH66336)); MEK and / or AKT kinase inhibitors. Inhibitors of cell signaling mediated by enzymes, c-kit inhibitors, abl kinase inhibitors, PI3 kinase inhibitors, Plt3 kinase inhibitors, CSF-1R kinase inhibitors, IGF receptor (insulin-like growth factor) kinase inhibitors; aurora kinase inhibitors (e.g., AZD1152, PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528 and AX39459) and cyclin-dependent kinase inhibitors such as CDK2 and / or CDK4 inhibitors; (v) Angiogenesis inhibitors such as agents that inhibit the action of vascular endothelial growth factor [e.g., the anti-vascular endothelial growth factor antibody bevacizumab (Avastin®) and, for example, VEGF receptor tyrosine kinase inhibitors, such as vandetanib (ZD6474), batalanib (PTK787), sunitinib (SU11248), axitinib (AG-013736), pazopanib (GW786034), and 4-(4-fluoro-2-methylindole-5-yloxy)-6-methoxy-7 Compounds such as (3-pyrrolidine-1-ylpropoxy)quinazoline (AZD2171; Example 240 in International Publication No. 00 / 47212), compounds disclosed in International Publication No. 97 / 22596, International Publication No. 97 / 30035, International Publication No. 97 / 32856 and International Publication No. 98 / 13354, and compounds that function by other mechanisms (e.g., linamides, inhibitors of integrin αvβ3 function, and angiostatins); (vi) vasoconstrictors such as combretastatin A4 and compounds disclosed in International Publication No. 99 / 02166, International Publication No. 00 / 40529, International Publication No. 00 / 41669, International Publication No. 01 / 92224, International Publication No. 02 / 04434 and International Publication No. 02 / 08213; (vii) Endothelin receptor antagonists, such as zibotentan (ZD4054) or atrasentan; (viii) Antisense therapies, such as ISIS2503 and anti-ras antisense therapies directed at the above targets; (ix) Gene therapy approaches including, for example, approaches that replace abnormal genes such as abnormal p53 or abnormal BRCA1 or BRCA2, GDEPT (gene-directed enzyme prodrug therapy) approaches, such as approaches using cytosine deaminase, thymidine kinase or bacterial nitroreductase enzymes, and approaches that enhance a patient's resistance to chemotherapy or radiotherapy, such as multidrug resistance gene therapy; and (x) Immunotherapy approaches, for example, ex vivo and in vivo approaches that enhance the immune response of a patient's tumor cells, including transfection of cytokines such as interleukin-2, interleukin-4, or granulocyte-macrophage colony-stimulating factor; approaches that reduce T cell anergy; approaches using transfected immune cells such as cytokine-transfected dendritic cells; approaches using cytokine-transfected tumor cell lines; and approaches using anti-idiotype antibodies; It may include one or more of the following.

[0177] In a particular embodiment, the growth inhibitory treatment as defined above may include, in addition to the compounds of the present invention, conventional surgery or radiotherapy or chemotherapy, wherein the chemotherapy may include one or more antitumor agents selected from cyclophosphamide, epirubicin, fluorouracil, methotrexate, mitomycin C, doxorubicin, gemcitabine, docetaxel, cabazitaxel and radium-223 dichloride.

[0178] In another specific embodiment, the growth inhibitory treatment defined above may include, in addition to the compounds of the present invention, conventional surgery or radiotherapy or chemotherapy, wherein the chemotherapy includes selective estrogen receptor modulators (SERMs) (e.g., tamoxifen or toremifene), aromatase inhibitors (AIs) (e.g., anastrozole, fadrozole, letrozole or exemestane), selective estrogen receptor degraders (SERDs) (e.g., fulvestrant, elacestrant or GDC-0810), and corpus luteum The antihormone agent may include one or more antihormone agents selected from the following: forming hormone (LH) blockers (e.g., goserelin), direct androgen receptor (AR) antagonists (e.g., bicalutamide, enzalutamide, apalutamide, darolutamide, cyproterone acetate, or flutamide), non-competitive AR antagonists (e.g., laraniten acetate), androgen steroid synthesis inhibitors (e.g., abiraterone acetate), and gonadotropin-releasing hormone (GNRH) modifiers (e.g., leuprolide, goserelin, buserelin, triptorelin, degarelix).

[0179] In another specific embodiment, the growth inhibitory treatment as defined above may include, in addition to the compounds of the present invention, conventional surgery or radiotherapy or chemotherapy, wherein the chemotherapy may include one or more cell cycle agents selected from cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitors (e.g., palbociclib, ribociclib, or abemaciclib).

[0180] In another specific embodiment, the growth inhibitory therapy defined above may include, in addition to the compounds of the present invention, conventional surgery or radiotherapy or chemotherapy, wherein the chemotherapy may include one or more DNA damage responders selected from poly-ADP-ribose polymerase (PARP) inhibitors (e.g., olaparib, veliparib, rucaparib, or niraparib).

[0181] In another specific embodiment, the growth inhibitory treatment defined above may include, in addition to the compounds of the present invention, conventional surgery or radiotherapy or chemotherapy, wherein the chemotherapy is a phosphatidylinositol 3 kinase (PI3K) inhibitor (e.g., buparisib, apitricib, azd8186, omiparisib, duvelisib, gedatolisib, copanlisib, pictilisib, alpelisib, idelalisib, acalisib, ceravelicib, pilaralisib, or ta The product may include one or more cell signaling agents selected from the following: taselisib, AKT inhibitors (e.g., MK2206, AZD5363, afrecertib, AT13148, miranceritib, or ipatasertib); (mTOR) signaling pathway inhibitors (e.g., everolimus, sirolimus, temsirolimus, vistuserti, sapanicetib, or ridaforolimus); and fibroblast growth factor (FGF) signaling inhibitors (e.g., AZD4547, or dovitinib).

[0182] In a particular embodiment, the growth inhibitory treatment as defined above may include, in addition to the compounds of the present invention, conventional surgery or radiotherapy or chemotherapy, wherein the chemotherapy may include one or more antitumor agents selected from procarbazine, carmustine, lomustine, irinotecan, temozolomide, cisplatin, carboplatin, methotrexate, etoposide, cyclophosphamide, ifosfamide, and vincristine.

[0183] In another specific embodiment, the growth inhibitory treatment as defined above may include, in addition to the compounds of the present invention, conventional surgery or radiotherapy or chemotherapy, wherein the chemotherapy may include one or more chemotherapeutic agents selected from BCL-2 family inhibitors (e.g., venetoclax and / or navigationoclax), BTK inhibitors (e.g., ibrutinib, acalabrutinib, tirabrutinib (ONO / GS-4059), BGB-3111 or spebrutinib (CC-292)), or TNF inhibitors (e.g., lenalidomide).

[0184] Such conjoint therapy can be achieved by administering the individual components of the treatment simultaneously, sequentially, or individually. Such combination products may include the compound of the present invention within the aforementioned dosage range and other pharmaceutically active agents within their approved dosage range.

[0185] According to this aspect of the present invention, a combination for use in the treatment of cancer (for example, cancer including solid tumors) is provided, comprising a compound of the present invention as defined above, or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof, and another antitumor agent.

[0186] According to this aspect of the present invention, a combination is provided for use in treating proliferative conditions such as cancer (e.g., cancer including solid tumors), comprising a compound of the present invention as defined above, or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof, and any one of the antitumor agents described herein.

[0187] In a further embodiment of the present invention, compounds of the present invention, or pharmaceutically acceptable salts, aqueous compounds, or solvated compounds thereof, are provided for use in the treatment of cancer in combination with another antitumor agent optionally selected from the above antitumor agents as described herein.

[0188] Where the term “combination” is used herein, it should be understood that it means simultaneous administration, individual administration, or sequential administration. In one aspect of the present invention, “combination” means simultaneous administration. In another aspect of the present invention, “combination” means individual administration. In a further aspect of the present invention, “combination” means individual sequential administration. When the administration is sequential or individual, any delay in the administration of the second component does not impair the beneficial effect of the combination.

[0189] A further aspect of the present invention provides a pharmaceutical composition comprising the compound of the present invention, or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof, together with an antitumor agent (optionally selected from the above antitumor agents as described herein), together with a pharmaceutically acceptable diluent or carrier. [Brief explanation of the drawing]

[0190] [Figure 1]A cSPR chemical library of LMO2-binding compounds is shown. (A) The SPR streptavidin chips used for cSPR screening were: channel 1: blank; channel 2: LMO2-ΔLID; channel 3: KRAS; channel 4: LMO2-GS-iDAb. Competitive SPR screening of the PPI-NET compound library (1,500 compounds in total) was performed for each compound at a single concentration of 150 μM (Cruz-Migoni et al., 2019). The responses measured for two control reference proteins: LMO2-iDAb, LMO2 fusion protein, or KRAS were normalized by subtraction to obtain a normalized response unit (RUnorm). Hit compounds Abd-L1, L2, L3, and L4 are shown as orange dots. Both LMO2-iDAb and KRAS were used as negative reference proteins. (C) The chemical structures and molecular weights (MW) of the four hits are shown, along with their PPI-NET plate position (P number) and designated antibody origin (Abd) number. Abd-L1 and Abd-L2 are homologs that differ only in the presence of 1H-pyrrolo[2,3-b]pyridine in Abd-L1 or pyrazolo[1,5-a]pyridine in Abd-L2, both indicated by red ovals. Note: Additional amounts of Abd-L2 and Abd-L3 are not commercially available. (D) The LMO2-binding hit compound Abd-L1 was evaluated in vitro using NMR waterLOGSY for binding to LMO2-LID or LMO2-ΔLID, compared to the compound's proton NMR spectrum. (E) Caco-2 permeability assay showing cell import and export data for Abd-L1. See also related Figure 7. [Figure 2]This paper demonstrates the establishment of a BRET-based LMO2-iDAb biosensor applicable to high-throughput screening of small molecule libraries. The BRET2 assay involves the live intracellular production of a signal following the interaction of a donor protein (in this case, LMO2-RLuc8) and an acceptor protein (in this case, GFP2-anti-LMO2 iDAb), and the BRET signal (energy transfer from activated RLuc8 to GFP2). (A) BRET donor saturation assay with donor LMO2 and different mutant iDAb LMO2 acceptors, iDAb LMO2dm, LMO2dm1~dm6. (B) BRET maxima and BRET 50 values ​​from the donor saturation curve represented in A. (C) Western blots for the expression of GFP2-iDAb LMO2 and mutants (using anti-GFP antibody), and LMO2-RLuc8 (using anti-LMO2 antibody). α-tubulin is the loading control. (D) BRET competition assay of LMO2-RLuc8 and different GFP2-iDAb LMO2dmx using either an unrelated control iDAb (anti-RAS (Tanaka et al., 2007); Ctl, white bar) or unmutated iDAb LMO2 (blank bar) as the competing substance. This competition is performed with the lowest dose of the competing substance (i.e., 0.1 μg). The percentage of inhibition by iDAb LMO2 compared to iDAb Ctl is shown. iDAb LMO2dm3 variants selected for cell-based screening assays are shown in blue. Each experiment was performed twice. If error bars are represented as (A,D), they correspond to the mean ± standard deviation (SD) of the biological repeat. See also related Figures 8-10. [Figure 3]This shows a cell-based high-throughput screening for inhibitors of LMO2-iDAb LMO2 PPIs. (A) A scheme of cell-based high-throughput screening (HTS) in which a diverse chemical library of 10,720 compounds was screened using a BRET cell assay to determine the reduction of the signal generated by the interaction of LMO2-RLuc8 and GFP2-iDAbdm3. (B) Scatter plot of normalized BRET signals from 10,720 compounds tested at 10 μM. 34 compounds (primary hits) caused inhibition of BRET signaling to less than 3 times the SD (minus, -3 × SD) cutoff of the DMSO BRET signal (Lavoie et al., 2013). Some primary hits are pinpointed in orange. (C,D) Confirmation of inhibition of BRET signaling from the interaction of iDAb LMO2dm3 with LMO2 (C) and the interaction of unmutated iDAb with LMO2 (D). The eight hits (shown by blue bars) were confirmed to reduce the LMO2-iDAb LMO2dm3 signal by at least 3×SD of the DMSO control BRET signal (i.e., DMSO BRET signal ±3×SD: 30.3±3.4, 26.9, threshold shown by dashed line) without affecting the LMO2-iDAb LMO2 interaction (i.e., DMSO BRET signal ±3×SD: 12.2±3~6, 8.6, threshold shown by wavy line). The P24H7 compound highlighted with a red asterisk is an example of a compound that was not further investigated because it affects both the iDAb LMO2dm3 interaction and the iDAb LMO2 interaction with LMO2. Experiments in C and D were performed twice. Error bars shown in C and D correspond to the mean ±SD of the biological replicates. See also related Figure 11. [Figure 4]This study demonstrates the structure-activity relationship of antibody-derived (Abd) LMO2-binding compounds. The chemical structures of hits from a BRET screen were investigated, and a family of compounds was identified. (A) Chemical structures of eight resynthesis hits (Abd-L5 to Abd-L12) with their respective molecular weights (MW). (B) Dose-response inhibition of LMO2-iDAb LMO2dm3 interaction by compounds Abd-L5 to Abd-L12 (concentration range: 1, 10, 20 μM). Data were obtained from duplicate biological experiments. Error bars correspond to the mean ± SD of the biological replicates. (C) SAR study of Abd-L9 compounds as templates. To obtain new compounds, the compounds were classified into four substituents substituted with various other chemical groups (named A to D). (D) Structures of representative compounds Abd-L15-L23. The novel compounds were tested by a BRET assay for LMO2-iDAb LMO2dm3 interaction. Different SAR-derived compounds are shown, along with their molecular weight and the percentage of BRET inhibition of LMO2-iDAb LMO2dm3 interaction at 20 μM. See also related Figures 12 and 13. [Figure 5](A-C) In vitro binding of Abd compounds to LMO2. In vitro binding affinity of Abd-L compounds confirmed by waterLOGSY NMR and photoaffinity labeling. (A-C) WaterLOGSY NMR was performed to determine the binding affinity of Abd-L9 (A), Abd-L10 (B), and Abd-L13 (C) to LMO2 fused to the LID of LDB1, or to the shortened version of the LID in LMO2-ΔLID. Each of these compounds binds to the LMO2-ΔLID (green) protein but not to the LMO2-LID (purple) protein. (D) Chemical structure of Abd-L26 designed for photoaffinity labeling (PAL) to the LMO2 protein. This is a compound constructed from the Abd-L15 / 16 template, possessing a benzophenone photoreactive site, a linker, and a biotin tag. (E-G) Pulldown of scFv-LMO2 recombinant protein with Abd-L26 using UV-treated or untreated avidin beads. Without UV treatment, the protein was incubated alone or with 20 μM Abd-L26 (lane 2). The protein was also incubated with 100 μM Abd-L9 as 20 μM Abd-L26 alone (lane 3) or as a UV-treated competitor (lane 4). The beads were washed, and the amount of cross-linked LMO2 on the beads was indicated from Western blot using anti-biotin (E), anti-LMO2 (F), and anti-HIS antibodies (G). [Figure 6]This report describes the evaluation of the titer of LMO2-binding Abd compounds in cells. The titer and specificity of LMO2 Abd compounds were evaluated using dose-response BRET assays (A-C). In the BRET assays, Abd-L9, Abd-L10, Abd-L16, and Abd-L22 compounds were evaluated by dose-inhibition responses to (A) LMO2-iDAb LMO2dm3, (B) LMO2-LDB1, and (C) LMO2-iDAb LMO2 (unmutated iDAb) BRET interactions. (D) Dose-response assays of Abd-L15, Abd-L17, Abd-L18, and Abd-L19 using LMO2-iDAb LMO2dm3, LMO2-LDB1, and LMO2-iDAb LMO2 BRET interactions. Each experiment was performed twice. The error bars shown in A-D correspond to the mean ± SD of the biological replicates. See also Figure 14. [Figure 7] In relation to Figure 1, molecular models of recombinant proteins used in cSPR library screening are shown. (A) LMO2-iDAb LMO2 (PDB ID: 4KFZ), (B) LMO2 only, (C) LMO2-ΔLID, and (D) LMO2-LDB1 LID domain fusion protein. LMO2 is shown as a surface presentation, the LDB1 LID domain as a rod, and iDAb LMO as a ribbon-like context, with the wavy line representing the linker between LMO2 and iDAb. The structures of LMO2 only, LMO2-LID, and LMO2-ΔLID are based on the LMO2-LDB1 crystal structure (PDB ID: 2XJY). The N-terminus and C-terminus of the proteins are marked for orientation. [Figure 8]Figure 2 shows the establishment of a BRET-based biosensor. (A) BRET donor saturation assay using LMO2-RLuc8 as the donor and GFP2-iDAb LMO2, GFP2-iDAb LMO2dm, and GFP2-iDAb Ctl (unrelated anti-RAS iDAb) as acceptors. (B) BRET competition assay between LMO2-iDAb LMO2 interactions when either iDAb Ctl (gray bar) or iDAb LMO2 (black bar) is used as the competitor (control non-competitive substance (-) is a white bar). (C) BRET competition assay between LMO2-iDAb LMO2dm interaction and the same competitor as in Panel B. (D) Western blot analysis of proteins from BRET competition assay cells shown in Panel C. Anti-GFP antibody shows iDAb LMO2dm expression and anti-LMO2 expression of LMO2-RLuc8, and anti-CMYC antibody shows expression of the competitor. Each experiment was performed twice. If error bars are shown, they correspond to the mean ± SD of the biological replicates. [Figure 9] In relation to Figure 2, the localization of iDAb LMO2 mutations on the LMO2 structure and the corresponding affected amino acids are shown. The LMO2 region around the hinge area is shown in gray and in each panel, and the relevant amino acids that interact with iDAb LMO2 are shown in red. (A) The localization of iDAb LMO2dm mutations (S55A and T107A) is shown in yellow on the parent iDAb LMO2 structure (cyan), and the affected interacting LMO2 residue (R109) is shown in red on the LMO2 structure. (B) The localization of iDAb LMO2dm3 mutations (S28G, H31G, S55A, E102A and T107A) is shown in yellow on the parent iDAb LMO2, and the affected LMO2 residue is shown in red on the LMO2 structure. (C) The localization of the iDAb LMO2dm6 mutation (S28G, H31G, S55A, E102A, S103A, and T107A) is shown in yellow on the parent iDAb LMO2 structure, and the affected LMO2 residues are shown in red on the LMO2 structure. The LMO2-iDAb LMO2 structure used is PDB 4KFZ. Each panel contains a list of interacting amino acids for LMO2 and iDAb. [Figure 10]Related to Figure 2, the DNA and protein sequences of iDAb LMO2 and its mutants are shown. (A) DNA and protein sequence of iDAb LMO2. (B-H) DNA and protein sequences of each iDAb LMO2dm1-LMO2dm6. Mutant amino acids compared to the parent iDAb LMO2 are indicated by a brown underline. [Figure 11] Related to Figure 3, BRET-based HTS control interactions are shown. (A) Signal controls of GFP2 only and RLuc8 only from Figure 3C. (B) Signal controls of GFP2 only and RLuc8 only from BRET experiments shown in Figure 3D. Compound-modified RLuc8 luminescence or endogenous GFP2 fluorescence was disposed of by more than 2 times (limit determined by the calculated dashed line relative to the DMSO control) (Lavoie et al., 2013). (C, D) Chemical structures of eight confirmed hits from HTS, divided into two related subfamilies, 5-membered ring (C) and 7-membered ring (D). (E) BRET competition assay with the eight hits and the unrelated PPI MAX bHLH-CMYC bHLH. Each experiment in (A, B, E) was performed twice. Error bars correspond to the mean ± SD of the biological replicates. [Figure 12]In relation to Figure 4, the anti-LMO2 Abd compounds are characterized. (A) Dose-response effects of Abd-L5~Abd-L12 compounds on LMO2-iDAb LMO2 (unmutated) interaction (Abd concentrations used: 1, 10, 20 μM). (B) Chemical structures and molecular weights (MW) of Abd-L13 and Abd-L14, two analogues of Abd-L8 and Abd-L21, respectively. (C) Abd-L13 and Abd-L14 were tested by BRET assays on LMO2-iDAb LMO2dm3 or (D) LMO2-iDAb LMO2 interaction (Abd concentrations used: 1, 10, 20 μM). (E) Dose-response effects of Abd-L15~Abd-L25 compounds on LMO2-iDAb LMO2dm3 interaction (Abd concentrations used: 5, 10, 20 μM). (F) Chemical structures of Abd-L24 and Abd-L25, with their respective molecular weights (MW) and their percentage of BRET inhibition of LMO2-iDAb LMO2dm3 interaction at 20 μM. These are two analogues modified at positions B and C, respectively, and do not affect the BRET interaction of LMO2-iDAb LMO2dm3. Experiments in A, C, D, and E were performed twice. Error bars shown in A, C, D, and E correspond to the mean ± SD of the biological replicates. [Figure 13] In relation to Figure 4, permeability assays of anti-LMO2 Abd compounds are shown. (A) Parallel artificial membrane permeability assay (PAMPA) with Abd-L9 and Abd-L16-18. (B) CaCo-2 permeability assay with Abd-L9 compound. [Figure 14]In relation to Figure 6, the establishment of a BRET-based LMO2-natural partner biosensor is shown. (A) BRET donor saturation assay using RLuc8-LMO2 as the donor and full-length TAL1-GFP2 as the acceptor, with or without co-expression of LDB1 and / or E47. BRET max and BRET 50 values ​​are shown for each BRET pair. (B) BRET donor saturation assay between LMO2-RLuc8 (donor) and full-length GFP2-LDB1 (acceptor). (C) BRET donor saturation assay using MAX bHLH-RLuc8 as the donor and CMYC bHLH-GFP2 as the acceptor. (D~F) The following interactions: (D) BRET competitive assays using iDAb Ctl (gray bar), iDAb LMO2 (black bar), or competitive substance (-, white bar) between LMO2-TAL1+E47, LMO2-LDB1, and MAX bHLH-CMYC bHLH. (G, H) BRET dose-response assays using LMO2-TAL1+E47 interaction (G) and MAX bHLH-CMYC bHLH interaction (H) with specified Abd-L compounds. Each experiment was performed twice. Error bars correspond to the mean ± SD of the biological replicates. [Examples]

[0191] The following examples are provided merely to illustrate the present invention as described herein and are not intended to limit the scope of the invention.

[0192] <abbreviation> Abd antibody-derived AITL Angioimmunoblastic T-cell lymphoma ALL Acute Lymphoblastic Leukemia AR androgen receptor BL Burkitt lymphoma BME 2-mercaptoethanol Boc t-butyloxycarbonyl BRET Bioluminescence Resonance Energy Transfer CML (Chronic Myeloid Leukemia) cSPR competitive surface plasmon resonance DLBCL (Diffuse Large B-cell Lymphoma) DMF (N,N-dimethylformamide) DMSO (Dimethyl Sulfoxide) EDTA (Ethylenediaminetetraacetic acid) ESI Electrospray Ionization HCl ethyl acetate FBS (Fetal Bovine Serum) FL (Follicular Lymphoma) GFP (Green Fluorescent Protein) GNRH (Gonadotropin-releasing hormone) GS Glycine Serine h time HATU N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide HBSS Hanks solution HPLC (High-Performance Liquid Chromatography) HPLC (High-Pressure Liquid Chromatography). HRMS high resolution mass spectrometry HTS High-Throughput Screening iDAb intracellular domain antibody IPTG Isopropyl 1-thio-β-D-galactopyranoside LB Lysogenic Broth LC-MS Liquid Chromatography-Mass Spectrometry LH (Luteinizing Hormone) LMO2 LIM domain-only protein 2 LRMS low resolution mass spectrometry mCPBA (m-chloroperoxybenzoic acid) MI Molecular Ions Minutes NMR nuclear magnetic resonance NSCLC (Non-Small Cell Lung Cancer) PAL photoaffinity labeling PAMPA parallel artificial membrane permeability assay Papp apparent transparency PARP (Poly-ADP-ribose polymerase) PBS (phosphate-buffered saline) PCR (polymerase chain reaction) PEG polyethylene glycol PPI protein-protein interactions ppm parts per million PS Penicillin / Streptomycin PVDF (Polyvinylidene Fluoride) RT room temperature RU (Responding Unit) SAR Structure-Activity Relationship SCC (Squamous Cell Carcinoma) SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) SERM (Selective Estrogen Receptor Degrader) SPR Surface Plasmon Resonance TFA (Trifluoroacetic Acid) THF (Tetrahydrofuran) TLC (Thin-Layer Chromatography)

[0193] <Analysis method> cell culture HEK293T cells were grown in DMEM medium (Life Technologies) and supplemented with 10% FBS (Sigma) and 1% penicillin / streptomycin (PS) (Life Technologies). Cells were grown at 37°C and 5% CO2.

[0194] Molecular cloning The DNA sequences encoding LMO2-ΔLID (UniProt P25791; residues 26-156 and UniProt Q86U70; residues 334-344, linked by a GS linker) and LMO2-iDAb were cloned into the expression vector pOPINS via restriction sites KpnI and HindIII, with AviTag incorporated into the 5' primer. The vector encodes an N-terminal hexa-histidine tag and a SUMO tag. The final protein expression construct encoded a single fusion protein consisting of His-SUMO-Avi-LMO2-GS-ΔLID and His-SUMO-Avi-LMO2-GS-iDAb. KRAS has an N-terminal His tag and a TEV protease cleavage site. G12V 166 The construct encoding the protein was modified via PCR to include an AviTag between the TEV site and the protein-coding sequence. For SPR screening, all proteins were prepared using biotin tags.

[0195] pEF-GFP as a template 2 -iDAb LMO2 dm We produced iDAb LMO2 mutations by PCR site-directed mutagenesis using (Bery et al., 2018) (i.e., iDAb LMO2 S55A / T107A). The following mutations were introduced: iDAb LMO2 S28G / H31G / S55A / T107A, iDAb LMO2 S55A / E102A / T107A, iDAb LMO2 S55A / E102A / S103A / T107A, iDAb LMO2 S28G / H31G / S55A / E102A / T107A, iDAb LMO2 S28G / H31G / S55A / S103A / T107A, and iDAb LMO2 S28G / H31G / S55A / E102A / S103A / T107A (Figure 10).

[0196] LMO2 cDNA was cloned into pEF-RLuc8-MCS and pEF-MCS-RLuc8 plasmids, and MAX bHLH (amino acids 37-102) was inserted into the pEF-MCS-RLuc8 plasmid. iDAb LMO2, mutant iDAb LMO2, and full-length LDB1 were converted to pEF-GFP. 2 - Cloned into MCS plasmid, full-length TAL1 and cMYC bHLH (amino acids 354-439) were converted to pEF-MCS-GFP. 2 It was cloned into a plasmid.

[0197] cSPR screening of PPI-NET compound libraries As previously described (Cruz-Migoni et al., 2019), biotin-labeled LMO2-ΔLID, KRAS, and LMO2-iDAb fusions were immobilized on streptavidin-coated sensor chips SA (GE Healthcare), and PPI-Net library compounds (containing 1,500 compounds) were injected onto its surface at a concentration of 150 μM. SPR experiments were performed as described using a Biacore T200 (GE Healthcare) at 10°C to preserve the proteins immobilized on the sensor surface. The control protein KRAS was used to obtain approximately equimolar immobilization levels of all three proteins on the sensor surface. G12V 166LMO2-iDAb was immobilized at approximately 4000 RU and 6000 RU, respectively. The flow cell was blocked by injecting 10 mM biocitin onto the surface at 10 μL / min for 5 minutes and used as a reference channel. Immobilization was performed in HEPES running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% P2O, 5 mM MgCl2, 10 μM ZnCl2). A compound solution was prepared by transferring 1.5 μL of 10 mM PPI-Net stock compound in 100% DMSO to a 96-well plate (Greiner) using a multi-channel pipette. 98.5 μL of running buffer containing 3.5% DMSO was added to obtain a 150 μM solution of the compound in running buffer containing 5% DMSO. The compound solution was injected into all four flow channels at 30 μL / min for 30 seconds, and dissociation was monitored for 60 seconds. After every 24-hour cycle, a negative control in running buffer containing 5% DMSO was run. Data were referenced, solvent corrected, and processed using T200 evaluation software. Data were baseline-corrected using the negative control binding level and the binding level measured for LMO2-ΔLID plotted against the binding level measured for the control protein as reference. Alternatives Abd-L1 (PPI-NET identifier P20000560B9) and Abd-L4 (PPI-NET identifier P20000557F5) were purchased from Asinex. Their molecular weights were confirmed by mass spectrometry (MS) and recorded using an Agilent 6120 spectrometer with a solution of MeOH: Abd-L1 LRMS m / z (ESI + )435[M+H] + ;Abd-L2LRMS m / z(ESI + )437[M+H] + .

[0198] BRET2 titration curve and competitive assay For all BRET experiments (titration curves and competitive assays), 650,000 HEK293T were seeded into each well of a 6-well plate. After 24 hours at 37°C, cells were transfected with a total of 1–6 μg of DNA mix containing donor + acceptor ± competitor plasmids using Lipofectamine 2000 transfection reagent (Thermo-Fisher). In dose-response competition experiments, the competitor was transfected with the following amounts of DNA: 0.1; 0.5 and 1 μg. In single-dose competition experiments, the competitor was transfected with 0.1 μg of DNA. Cells were separated after 24 hours, washed with PBS, and seeded into white 96-well plates (clear bottom, PerkinElmer, catalog no. 005181) in OptiMEM phenol red-free medium supplemented with 4% FBS. Cells were incubated for a further 20–24 hours at 37°C before reading the BRET assay. A detailed BRET protocol is provided by Bery and Rabbittss, (2019).

[0199] Cell processing The compounds were prepared in 100% DMSO at 10 mM. For the BRET competitive assay, cells were treated with the specified compound at concentrations of 1 (or 5), 10, and 20 μM for 22 hours. For the BRET-based dose-response experiment, cells were treated with the compound at concentrations of 0.01, 0.1, 1, 4, 10, 25, and 50 μM for 22 hours. BRET medium: The compounds were diluted in OptiMEM phenol red-free medium (Life Technologies) supplemented with 4% FBS and having a final concentration of 0.2% DMSO.

[0200] BRET2 measurement Using a CLARIOstar instrument (BMG Labtech) equipped with a luminescence module, the BRET2 signal was determined immediately after injecting cells (Cayman Chemicals) with 400a substrate (final 10 μM). Total GFP was measured at excitation and emission peaks set at 405 nm and 515 nm, respectively. 2Fluorescence was detected. Total RLuc8 luminescence was measured using a luminescence 40-700 nm wavelength filter.

[0201] The BRET signal or BRET ratio was divided by the light emitted by the RLuc8 donor construct (410 nm ± 80), and when coelenterazine 400a was added, GFP was produced. 2 This corresponds to the light emitted by the acceptor construct (515 nm ± 30). The background signal is subtracted from the BRET ratio using a donor-only negative control when only the RLuc8 fusion plasmid is transfected into cells. The normalized BRET ratio is the BRET ratio normalized to the negative control (iDAb control or DMSO control) during the competitive assay. Total GFP 2 Protein expression from each plasmid was controlled using the RLuc8 signaling pathway.

[0202] Western blot analysis Cells were washed once with PBS and lysed in SDS-Tris buffer (1% SDS, 10 mM Tris HCl pH 7.4) supplemented with a protease inhibitor (Sigma) and a phosphatase inhibitor (Thermo Fisher). Cell lysates were sonicated with a Brason Sonifier, and protein concentrations were determined using the Pierce BCA protein assay kit (Thermo Fisher). Equal amounts of protein (20 μg) were separated on 12.5% ​​SDS-PAGE and subsequently transferred to a PVDF membrane (GE). The membrane was blocked with 10% skim milk (Sigma) in TBS-0.1% Tween20 and incubated overnight at 4°C with the primary antibody. After washing, the membrane was incubated with an HRP-conjugated secondary antibody at room temperature (RT, 22°C) for 1 hour. The membranes were washed with TBS-0.1% Tween and developed using Clarity Western ECL Substrate (Bio-Rad) and CL-XPosure film (Thermo Fisher) or ChemiDoc XRS+ imaging system (Bio-Rad). Primary antibodies included anti-LMO2 (1 / 1000, R&D System, catalog number AF2726), anti-GFP (1 / 500, Santa Cruz Biotechnologies, catalog number sc-9996), anti-biotin (1 / 1000, CST, catalog number 5597S), anti-β-actin (1 / 5000, Sigma, catalog number A1978), and anti-tubulin (1 / 2000, Abcam, catalog number ab4074). Examples of secondary antibodies include anti-CMYC HRP conjugate (Novus Biologicals, catalog number NB600-341), anti-mouse IgG HRP conjugate (CST), anti-rabbit IgG HRP conjugate (CST), and anti-goat IgG HRP conjugate secondary antibodies (Santa Cruz Biotechnologies).

[0203] High-throughput chemical screening using LMO2 / iDAb LMO2 mutant BRET biosensor Screening was performed in 384-well plate format. The in-house library of 10,720 compounds (including 6,991 compounds from BioFocus and 3,729 compounds from ChemBridge) was in 96-well plate format. For the purpose of HTS, the library was compressed into 384-well plate format. The specified volume and quantity are for 40 assay 384-well plates. Screening was performed in pairs at 10 μM. Two HTS sessions, each containing 5,360 compounds, were screened in 68 assay plates (34 assay plates in pairs).

[0204] Before starting, HEK293T cells were seeded in 2×T175 flasks. After 3 days, the 2×T175 flasks were divided into 6×T175 flasks.

[0205] Day 1: Cell Seeding: Cells were collected from 6×T175 flasks at approximately 70% confluence. The cells were resuspended in 110 mL of complete DMEM and placed in two Corning HyperFlasks (Corning, catalog no. 10030), each containing 120×10 cells. 6 Cells were inoculated, and 560 mL of culture medium was added to fill one hyperflask.

[0206] Day 2: pEF-LMO2-RLuc8 and pEF-GFP 2 -iDAb LMO2 dm3 Cell transfection: In each hyperflask, add 10 mL of OptiMEM, 19 μg of pEF-LMO2-RLuc8, and pEF-GFP. 2 -iDAb LMO2 dm337 μg of the DNA was added along with 244 μg of the pEF-empty-cyto-myc plasmid. 750 μL of Lipofectamine 2000 was added in 10 mL of OptiMEM and gently mixed. 10 mL of DNA diluent was added and incubated for 20 minutes. The DNA / Lipofectamine 2000 mixture was added to 500 mL of complete DMEM, and the medium in the hyperflask was removed. Finally, the medium + transfection mixture was carefully poured into the hyperflask without creating bubbles, and the flask was filled with medium.

[0207] Day 3: Cell seeding in 384-well plates: Cells were collected using 100 mL of trypsin added to each hyperflask, incubated at 37°C for 2 minutes, and transferred to a beaker containing 100 mL of complete DMEM. Each flask was washed once with 100 mL of complete DMEM and gently but thoroughly mixed to ensure the formation of a single-cell suspension (final volume of one flask: 300 mL). 250 mL Corning centrifuge tubes (total 360 × 10⁶) 6 Each transfected cell sample (using 4 x 250 mL centrifuge tubes) yielded 90 x 10 cells per tube. 6 Cells were added and centrifuged at 220 × g for 5 minutes at room temperature. Each cell pellet (4 cells in total) was gently resuspended in 200 mL of Opti-MEM (hereafter referred to as BRET medium) containing 4% FBS + 1% PS and without phenol red, to a final concentration of 0.45 × 10⁶. 6 Cells were seeded into a white 384-well plate (clear bottom, PerkinElmer, catalog no. 6007480) using a PerkinElmer Janus liquid handling workstation housed in a Category 2 sealed container (45 μL / well; 20,000 cells). A blank plate was used first to remove air bubbles in the liquid handling workstation.

[0208] Day 3: Library Dilution: A 100 μM stock solution was prepared for each compound in the library (the initial concentration of the library was 10 mM). Using an Echo Acoustic dispenser (Labcyte), 150 nL of each compound from the 10 mM solution was added to 15 μL of BRET medium to obtain a final concentration of 100 μM.

[0209] Day 3: Compound addition: 1% DMSO was prepared in BRET medium. As a negative control, 5 μL of 1% DMSO solution was partitioned into columns 1, 2, 23, and 24. Using a PerkinElmer Janus liquid handling workstation, 5 μL (100 μM) of the compound was added to the cells in each well (final concentration 10 μM, 0.1% DMSO), and the plates were incubated for 20 hours.

[0210] Day 4: Plate Reading: Plates equipped with a BRET2 optical module were read using a PHERAstar FSX plate reader (BMG Labtech). GFP of each plate was read. 2 The signal was measured first to assess the relative cell count in each well. GFP 2 After reading the BRET signals, the bottom of the plate was covered with white tape. 80 mL of 100 μM BRET substrate (i.e., coelenterazine 400a, Cayman Chemicals) was prepared by dissolving 3 mg of coelenterazine 400a in 32 mL of 100% ethanol, and 48 mL of BRET medium was added to bring the volume to 80 mL. BRET readings were performed by adding 5.5 μL of coelenterazine 400a (final concentration 10 μM) using an injector and reading the BRET signal from each well. The reading time for one 384-well plate was approximately 8 minutes, and therefore approximately 4.5 hours for reading 34 plates.

[0211] Recombinant protein expression LMO2-LID protein was expressed in BL21(DE3)-Rosetta2 pLysS cells (Novagen), and HIS-SUMO-AVI-LMO2-ΔLID was expressed in Lemo21(DE3) cells (NEB, catalog number 2528J). Bacterial cells were cultured at 37°C in LB medium supplemented with 50 μg / mL kanamycin and 32 μg / mL chloramphenicol. Expression was induced by adding IPTG supplemented with final concentrations of 0.5 mM and 0.1 mM ZnCl2 during the logarithmic phase. The cultures were incubated overnight at 18°C ​​with shaking at 220 rpm. Cells were collected by centrifugation and resuspended in binding buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 5% glycerol, and 5 mM imidazole). The resuspended pellet was stored at -20°C. The thawed cell pellet was supplemented with a 1× concentration (Roche) of a completely EDTA-free protease inhibitor at 5 μL DNAse / culture volume (L) and MgSO4 (final concentration 2 mM). The cell suspension was stirred on ice for 15 minutes and lysed at 23 kpsi, 4°C using a Constant Systems Cell Disruptor. The cell extract was clarified by centrifugation. Histag proteins were purified under gravity flow using nickel-Sepharose (GE Healthcare). The bound proteins were washed with 2 × 50 mL of binding buffer, followed by washing with 25 mL of washing buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 5% glycerol, and 20 mM imidazole). The bound protein was eluted with 5 mL of elution buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 5% glycerol, and 50 mM imidazole) and elution buffer 2 (2 × 5 mL) (50 mM HEPES, pH 7.4, 500 mM NaCl, 5% glycerol, and 500 mM imidazole). The SUMO tag was removed by incubation overnight with SUMO protease at 4°C. The protein was further purified on a HiLoad 16 / 60 Superdex 200 Prep Grade column using an AKTA Avant system (GE Healthcare) buffered in 10 mM HEPES (pH 7.4) and 250 mM NaCl.For the proteins used in the SPR experiment, the N-terminal AviTag was biotin-labeled by incubation overnight at 4°C with BirA enzyme in the presence of 20 mM MgCl2, 500 μM biotin, and 2 mM ATP. The proteins were further purified on a HiLoad 16 / 60 Superdex 200 Prep Grade column using an AKTA Avant system (GE Healthcare) buffered in 10 mM HEPES (pH 7.4), 250 mM NaCl, and 0.5 mM DTT. The purified proteins were then biotin-labeled using an additional BirA incubation step as described above in KRAS. 166 G12V It was purified (Cruz-Migoni et al., 2019).

[0212] Purification of scFv-LMO2 for PAL analysis For the co-expression of recombinant LMO2 and anti-LMO2 scFv, scFv was cloned into an existing bicistronic expression vector (pRK-His-TEV-VH576-LMO2, Sewell et al 2014). The DNA encoding scFv was amplified by PCR, cloned into the pRK vector, and VH576 was replaced using the NcoI and EcoRI restriction sites. For co-expression of the proteins, Escherichia coli (E. coli) C41 (DE3) cells were transformed with the plasmid DNA. Using single colonies, LB medium containing 100 μg / mL ampicillin was inoculated and grown overnight at 37°C with shaking at 225 rpm. The overnight seed culture was diluted 1:100 in 8 x 1L LB mediums containing 100 μg / mL ampicillin. 600The culture was grown at 37°C with shaking at 225 rpm until it reached 0.6. ZnSO4 was added to a final concentration of 0.1 mM before induction. Protein expression was induced by adding 0.5 mM IPTG (isopropyl 1-thio-β-D-galactopyranoside), and the cells were incubated at 16°C with shaking at 225 rpm. Cells were collected by centrifugation at 6000 rpm at 4°C for 20 minutes. The cell pellet was resuspended in a lysis buffer (20 mM Tris (pH 8.0), 250 mM NaCl, 20 mM imidazole, 0.1 mM ZnSO4, 5 mM 2-mercaptoethanol, 5% glycerol) containing an EDTA-free protease inhibitor cocktail tablet (Roche, Germany) before lysis at 25 kPSI at 4°C using a cell disruption system (Constant Systems Ltd, UK). Before clarification by centrifugation at 2,000 rpm at 4°C for 1 hour, the cell lysates were incubated with DNase I and 2 mM MgCl2 at room temperature for 20 minutes. LMO2 and anti-LMO2 scFv were simultaneously purified using a 5 mL HisTrap HP column (GE Healthcare, UK) with a 50 mL imidazole gradient of 20 mM to 300 mM. The proteins were concentrated to 1.5 mL and further purified by filtration using a HiLoad 16 / 600 Superdex 75 column (GE Healthcare, UK) in 20 mM Tris (pH 8.0), 250 mM NaCl, and 1 mM DTT. Simultaneous purification of LMO2 and anti-LMO2 scFv was verified by standard Western blotting using anti-LMO2 (R&D Systems, AF2726) and anti-His-HRP (Sigma, A7058).

[0213] WaterLOGSY NMR LMO2-ligand interactions were measured using WaterLOGSY NMR (Bataille et al., 2020). WaterLOGSY experiments were performed at a 1H frequency of 600 MHz using a Bruker Avance spectrometer equipped with a BBI probe. All experiments were performed at room temperature. A 3 mm diameter NMR tube with a sample volume of 200 μL was used for all experiments. For LMO2-LID, the solution was buffered with a 10 mM NaPO4, 250 mM NaCl solution. For LMO2-ΔLID, the solution was buffered with a 10 mM NaPO4, 50 mM NaCl solution. Sample preparation for measuring ligand binding affinity to LMO2-LID is exemplified below: the compound (10 μL of 10 mM solution in DMSO-d6) was added to an Eppendorf tube before sequentially adding the appropriate buffer (90 μL), D2O (20 μL), and protein (80 μL, 25 μM). Before NMR analysis, the obtained solution was vortexed and mixed, then transferred to a 3 mm NMR tube. Sample preparation for measuring ligand binding with LMO2-ΔLID is exemplified below: the compound (10 mM solution in DMSO-d6, 10 μL) was added to an Eppendorf tube before sequentially adding a suitable buffer (162 μL), D2O (20 μL), and protein (8 μL, 250 μM). Before NMR analysis, the obtained solution was vortexed and mixed, then transferred to a 3 mm NMR tube. Sample preparation for confirming possible aggregation with ligand only is exemplified below: the compound (10 mM solution in DMSO-d6, 10 μL) was added to an Eppendorf tube before sequentially adding a suitable buffer (170 μL) and D2O (20 μL). Before NMR analysis, the obtained solution was vortexed and mixed, then transferred to a 3 mm NMR tube.

[0214] PAL pulldown To a final volume of 400 μL of PBS containing 40 μg of the target purified protein, 20 μM of Abd-L26, either containing or not containing 100 μM of Abd-L9 (competitor), was added. The same sample was prepared for a control without UV treatment. The samples were incubated at room temperature for 25 minutes. The samples to be crosslinked were placed on ice and left under a UV lamp for 1 hour for crosslinking. The control without UV treatment was kept on ice. During the 1 hour crosslinking period, agarose monomer avidin beads (catalog number 20228, Thermo Fisher) were washed twice with PBS. After crosslinking, 20 μL of the washed beads were added to all samples (crosslinked and uncrosslinked) and incubated on a roller at 4°C for 2 hours. After 2 hours, the beads were washed three times with 400 μL of PBS. Finally, the sample is denatured with 50 μL of 2× loading buffer, BME is added directly to the beads (and boiled at 100°C for 5 minutes), and then loaded for Western blot analysis.

[0215] CACO-2 and PAMPA assays As described (Bavetsias et al., 2016), the apparent permeability (Papp) of Caco-2 in the Caco-2 human colon cancer cell line was determined. Cells were maintained for 10 days in a humidified atmosphere of 5% CO2 / 95% air (DMEM containing 10% fetal bovine serum, penicillin, and streptomycin). Cells were plated onto cell culture assembly plates (Millipore, UK), and monolayer confluence was confirmed using a TEER electrode before assay. The medium was washed and replaced with HBSS buffer (pH 7.4) containing the compound (10 μM, 1% DMSO) in appropriate apical and basal donor wells. Only HBSS buffer was placed in the acceptor wells. In specific cases, the specific P-gp inhibitor, LY335979 (5 μM), was added to the HBSS. Caco-2 plates were incubated at 37°C for 2 hours. Samples from the apical and basal outer chambers were analyzed using the Waters (Milford, MA, US) TQ-S LC-MS / MS system. The cell permeability of Abd-L compounds was compared with low (nadolol) and high (antipyrine) permeability compounds and compounds with high transport (indinavir).

[0216] The apparent transparency (Papp) was determined as follows:

number

[0217] PAMPA The permeability of the compound was determined by passive diffusion using a parallel artificial membrane permeability assay (PAMPA). The assay used an artificial membrane consisting of 2% phosphatidylcholine in dodecane (Sigma Aldrich, Dorset, UK). The donor plate was a MultiScreen-IP Plate with 0.45 μM hydrophobically bound Immobilon-P Membrane (Millipore, UK), and the acceptor plate was a MultiScreen 96-well Transport Receiver Plate (Millipore, UK). Permeability was measured at three different pH levels: pH 5, 6.5, and pH 7.4 in a buffer containing 1% bovine serum albumin (Sigma Aldrich, Dorset, UK). A 10 mM DMSO stock solution of the test compound was used to create 10 μM PAMPA donor solutions and calibration curves for each of the three buffers.

[0218] 6 μL of membrane solution was added to each well of the donor plate. 200 μL of buffered donor solution was added to the appropriate wells of the PAMPA donor plate. 300 μL / well of blank PBS (pH 7.4) was added to the PAMPA acceptor plate.

[0219] Next, the donor plate and acceptor plate were sandwiched together, covered with a lid, and incubated in a humid environment at 30°C for 16 hours. After the incubation period, the plates were removed from the incubator and the sandwich was disassembled. The samples were then transferred to new plates and centrifuged. The supernatants of all samples were diluted and analyzed using a Waters (Milford, MA, US) TQ-S LC-MS / MS system.

[0220] The transmittance value (cm / s) was calculated using the following equation:

number

[0221] <Result> In vitro cSPR screening of chemical libraries containing LMO2-iDAb fusion proteins The use of high-affinity intracellular antibody conjugation to RAS proteins in competitive SPR screening of chemical library screens has been previously described (Quevedo et al., 2018), and relies on the high-affinity interaction between the antibody and antigen on the SPR chip to select Abd compounds. The same approach was employed for the conjugation of intracellular antibodies (in the form of iDAb) to the LMO2 protein. In this case, since the interaction affinity is in the nM range rather than pM for anti-RAS, a fusion of LMO2 and iDAb was used, with the two components linked in a single polypeptide by a short, mobile glycine-serine (GS) linker (Figure 7A compared with the structure of LMO2 from PDB 2XJY in Figure 7B). Since this has been found to be the only effective way to express the soluble LMO2 protein apart from co-expression with iDAb (Ryan et al., 2006), LMO2 can also be expressed in Escherichia coli (E. coli) in complex with the LID domain of LIM domain binding 1 (LDB1). The binding of iDAb to LMO2 occurs across the LMO2 hinge, which overlaps to some extent with the LDB1 LID binding (Sewell et al., 2014). As a result, a cleaved version of the LID domain is co-expressed with LMO2 (hereafter, LMO2-ΔLID), which covers the entire LIM2 region of LMO2 while leaving the accessible hinge and LIM1 region (Figure 7C compared with the structure of LMO2-LID from PDB 2XJY in Figure 7D). Therefore, the SPR chip was constructed with LMO2-iDAb in one channel, LMO2-ΔLID in the second channel, and the unrelated protein, KRAS, attached to the third channel. The chip format is shown in Figure 1A. This configuration of the SPR channel allowed for the subdivision of chemical substances into those that can bind to LMO2 in the iDAb-binding region (Abd compounds as iDAb substitutes), those that bind to LMO2 in other locations besides the LID and iDAb-binding region, and those that bind to the RAS.

[0222] Among 1,500 screened compounds, four LMO2-ΔLID Abd hits with more than 10 response units that did not bind to LMO2-iDAb or KRAS were identified (Figure 1B). Their chemical structures and molecular weights, along with response unit data from the screening, are shown in Figure 1C. Compounds designated Abd-L1 and Abd-L4 are commercially available, but Abd-L2 (an analogue of Abd-L1) and Abd-L3 were not. Further characterization of Abd-L1 was initiated using orthogonal assays with LMO2-LID and LMO2-ΔLID, and 1D NMR waterLOGSY (Dalvit et al., 2001, Bataille et al., 2020). While the proton peaks showed a polarity shift induced by interaction with LMO2-ΔLID, limited changes were observed with respect to the LMO2-LID protein (Figure 1D).

[0223] The objective of the compound library screening is to identify chemicals that can form the basis for drug development and therefore function in cells. Accordingly, the cellular permeability of Abd-L1 in the CaCo-2 assay was evaluated compared to compounds with low (nadolol) and high (antipyrine) permeability and high transport (indinavir) (Figure 1E). The CaCo-2 data indicates that Abd-L1 exhibits low cellular permeability with high efflux in CaCo-2 cells, suggesting insufficient cell-based drug properties (Figure 1E).

[0224] Establishment of a BRET-based LMO2-iDAb biosensor for small molecule screening. In vitro selection assays clearly do not always generate cell-permeable compounds; therefore, an alternative approach was specified using cell-based screening methods for iDAb substitutes to improve the cellular properties of the chemical hits (i.e., cell uptake, low transport, etc.). Such cell-based screening for compounds that inhibit PPIs requires assays that generate signals from PPIs that do not occur via high-affinity interactions, as initial chemical hits are expected to be weak binders. Thus, a BRET-based LMO2 / iDAb LMO2 biosensor was designed based on the RAS biosensor strategy (Bery et al., 2018). Using structural data of the LMO2-iDAb LMO2 complex (Sewell et al., 2014), the vicinity of the donor and acceptor sites was optimized. The donor site RLuc8 was fused to the carboxyl terminus of iDAb LMO2, and GFP 2 The acceptor molecule was fused to the amino terminus of iDAb LMO2. LMO2-RLuc8 and GFP 2 -iDAb interaction with LMO2, low affinity GFP 2 -iDAb LMO2 dm (Dematured iDAb LMO2 (Bery et al., 2018)) or unrelated GFP 2 -iDAb RAS (Tanaka et al., 2007) (hereinafter referred to as iDAb control or iDAb Ctl) was tested by BRET donor saturation assay (Figure 8A). From these data, iDAb LMO2 was compared to iDAb LMO2. dm BRET 50 Because there is a 10-fold increase in (approximate relative affinity of the acceptor to the donor protein) (0.44 vs. 0.03, respectively, see Figure 8A), dematuration mutagenesis leads to iDAb LMO2 dm It has been demonstrated that the affinity decreases. Untagged competing substance (iDAb LMO2) or unrelated competing substance (iDAb Ctl) is compared to BRET vs. LMO2-iDAb LMO2 (Figure 8B) or LMO2-iDAb LMO2. dmThe characteristics of these interactions were evaluated using a BRET competition assay, expressing either of the substances shown in Figure 8C. The competitor iDAb LMO2 showed dose-dependent reduction, but at the highest dose of the competitor it reduced to only about 65% (Figure 8B). Therefore, iDAb LMO2 showed stronger inhibition at its highest dose and lower affinity iDAb LMO2 dm - LMO2 interactions were made competitive (approximately 80%, Figure 8C), and the expression of these proteins remained unchanged (Figure 8D).

[0225] Using a dematuration method, iDAb affinity based on the CDR sequence is reduced (Assi et al., 2010), for example, RAS G12V Alpha-screening of binding compounds and analysis of in vitro RAS-binding Abd compounds have become possible (Tanaka & Rabbitts, manuscript in preparation). Based on LMO2-iDAb LMO2 structural information (Sewell et al., 2014), the inventors have developed iDAb LMO2 with alanine or glycine substitutions that will affect the interaction between the major amino acids from iDAb and LMO2 while still maintaining specific binding affinity. dm Further mutations were introduced on the CDR (Figure 9A-C). Most of the modifications on iDAb LMO2 affected its binding affinity around the hinge region of LMO2 (Figure 9A-C). iDAb LMO2 dm Six further variants, designated 1-6 (DNA and protein sequences shown in Figures 10A-H), were created and tested in a BRET donor saturation assay (Figure 2A). Each of the iDAb LMO2 variants (dm1-6) was compared to the template iDAb LMO2. dm Compared to, reduced BRET 最大 Values ​​(total number of LMO2 / iDAb complexes and approximate distance between donor and acceptor in the dimer) and increased BRET 50The value was obtained (Figure 2B). This suggests that the reduced overall affinity of the dematured iDAb for LMO2 and the mutation does not affect its expression (Figure 2C). Finally, BRET competition experiments were performed on each mutant using the highest dose of the competitor to determine the optimal dematured iDAb for chemical library screening. iDAb LMO2 dm3 Competitive data showed that this mutant was the best because its interaction with LMO2 was almost completely inhibited (approximately 90%) by iDAb LMO2 while maintaining a relatively high BRET signal (Figure 2D). Therefore, this mutant was selected for cell-based high-throughput screening of small molecules.

[0226] LMO2-iDAb dm3 High-throughput screening for interaction inhibitors Cell-based BRET LMO2-iDAb LMO2 dm3 The robustness and scalability of the interaction assay were tested using high-throughput screening (HTS) to identify compounds that inhibit this interaction. A library of 10,720 small molecules constructed from Biofocus and Chembridge sources was screened. The HTS flowchart is shown in Figure 3A. On day 1, HEK293T cells were injected with LMO2-RLuc8 and GFP. 2 -iDAb LMO2 dm3 Plasmids expressing were transfected, and after 24 hours, the compound was added to 10 μM, and the BRET signal was determined after another 24 hours. Using a 3×SD cutoff from the DMSO control, 34 primary hits were identified (Lavoie et al., 2013) (Figure 3B). These were retested using the original BRET assay to confirm signal inhibition and using a BRET-based interaction assay between LMO2 and unmutated iDAb LMO2 to rule out the binding affinity of the compound to iDAb (Figures 3C, D). Furthermore, RLuc8 luminescence or endogenous GFP were tested. 2Early hits that have more than a twofold impact on fluorescence were not taken into consideration (Figure 11A, B). As a result of this re-screening, LMO2-iDAb LMO2 dm3 Eight inhibitors of the interaction were obtained (Figure 11C, D). These eight compounds were finally tested using an unrelated BRET-based interaction assay (MAX bHLH-CMYC bHLH) to provide further confirmation of specific interactions with LMO2 (Figure 11E). From the chemical structures of the selected hits, it was found that the compounds belong to a family that can be divided into two subfamilies based on their chemical similarity. The main difference is the presence of a five-membered or seven-membered ring in each subfamily (Figure 11C, D, respectively).

[0227] Study of structure-activity relationships of Abd compounds Samples containing a five-membered ring (Figure 11C) were prepared according to the literature method. However, when attempting to synthesize samples of compounds containing a seven-membered ring, it was found that the core was subject to rearrangement to a five-membered ring (Figure 11D). Therefore, corresponding five-membered ring analogs, called Abd-L5 to Abd-L12, were synthesized (Figure 4A). These analogs were tested by dose-response BRET assays to verify their ability to bind to LMO2 and their titer (Figures 4B and 12A). Three of the analogs, Abd-L5, Abd-L8, and Abd-L11, were found to be LMO2-iDAb LMO2. dm3 The interaction could not be inhibited (Figure 4B). For structure-activity relationship studies, the most potent inhibitor, Abd-L9, was used as a template (Figures 4C, D).

[0228] Different sites of the Abd compounds were systematically modified by dividing them into four substituents: benzyl (position A), imidazolidinone (B), oxazole (C), and aniline (D) (Figure 4C). Representative analogues are shown (Abd-L13 to Abd-L25, Figure 4D and Figures 12B to F). Most substituted benzyl groups were found to be tolerable at position A, and methoxy groups can be placed at the α, meta, or para positions of the benzyl ring with minimal effect on the BRET inhibitory titer of the derivatives (compounds with benzyl modification (red box) and / or aniline modification (green box) are shown in Figure 4C). Large arrays of substituted anilines and benzylamines were also found to be tolerable at position D. It is noteworthy that most substituted pyridine-containing compounds did not exhibit activity resulting from changes in the basicity of the compound.

[0229] Modifications at positions B and C had a greater effect on the titer of the analogs. At position B, substitution of any imidazolidinone was found to decrease activity, apart from the corresponding piperazine (pink box in Figure 4D) (hence pyrimidinone and piperazine, Figures 12E, F). Due to the potential chemical instability, low yield, and numerous byproducts in the rigidity of imidazolidinone, the piperazine moiety was substituted for further SAR studies. These issues were resolved by modifying the piperazine. At position C, only 2,4-substituted thiazoles and 2,4-substituted oxazoles were found to be tolerable as cores, while the corresponding 2,5-substituted heterocycles and different heterocycles (such as pyrimidines) caused a decrease in activity (see blue box on position C of the analogs in Figures 4D and 12E, F). These data suggest that the B / C position is important for the interaction between the compound and LMO2, and that the A / D position can be modified to add new functional groups.

[0230] Abd-L9 and some analogues were tested in a parallel artificial membrane permeability assay (PAMPA), and Abd-L9 was tested in a Caco-2 permeability assay (Figure 13A, B). These results showed that, similar to expected compounds derived from cell-based screens, the compounds were permeable through synthetic membranes (PAMPA) or into cells (Caco-2). Abd-L9 exhibited the best properties in PAMPA compared to its analogues, and showed low transport but low efflux ratio in the Caco-2 assay (Figure 13A, B). These results suggest that Abd-L9 enters cells with relatively low efficiency but is not actively transported out of cells (low efflux ratio).

[0231] Abd compound binds to LMO2 in vitro. In cell-based assays, Abd compounds were identified and validated. LMO2-LID and LMO2-ΔLID were used in waterLOGSY NMR experiments to evaluate the binding affinity of LMO2 to small molecules. One compound from a cSPR screen (Abd-L1, Figure 1D) and three LMO2 compounds from cell-based screens were tested (Abd-L9, Abd-L10, and Abd-L13, Figures 5A-C), each showing binding affinity to LMO2-ΔLID rather than LMO2-LID. The differences between the two proteins are the hinge and LIM1, which is consistent with cell data indicating that the compounds bind to LMO2 and that their binding is limited to the interface between LIM1 and the hinge region of LMO2.

[0232] These data were further confirmed using an alternative method: photoaffinity labeling (PAL), a powerful technique used to investigate protein-ligand interactions (Smith and Collins, 2015). PAL involves the use of chemical probes that, upon photoactivation, can covalently bind to their target (Sadakane and Hatanaka, 2006). Extensive SAR data for LMO2 Abd compounds suggested a binding site on the parent ligand. A benzophenone photoreactive group was added to the benzyl substituent (position A), and a linker with a biotin tag was added at position D (Figure 5D). To obtain a soluble recombinant LMO2 protein with an accessible Abd-L compound-binding site, phage display screening of scFvs was performed with LMO2-LID protein antigen to obtain scFvs that could bind to LMO2 and be co-expressed in E. coli (Miller & Rabbitts, unpublished). By using a partially purified LMO2-scFv dimer, the PAL technique was performed after inducing crosslinking of a photoaffinity LMO2 PAL compound (specified Abd-L26; Figure 5D, containing a photoactive substituent and a biotin moiety linked to the compound by a short linker) to the scFv-LMO2 complex with ultraviolet light for photocrosslinking. Abd-L26 in the complex was isolated by the interaction of avidin beads and the biotin moiety, and the protein was analyzed by Western blotting with either an anti-biotin antibody (Figure 5E), an anti-LMO2 antibody (Figure 5F), or an anti-histag (Figure 5G). From the pull-down data, it was shown that the protein was only crosslinked when the mixture was treated with ultraviolet light, and the inventors confirmed the size of the LMO2 (Figure 5F) and the biotin-labeled protein (Figure 5E, lane 3) simultaneously. Furthermore, the recovery of the biotin-labeled LMO2 was inhibited by incubating the protein with Abd-L26 (PAL) in the presence of a 5-fold concentration of the Abd-L9 competitor (Figure 5E, lane 4). Anti-biotin antibodies revealed that the biotin-labeled protein specifically binds to the beads via the PAL Abd-L26 compound, while anti-LMO2 and anti-his antibodies show non-specific binding of the protein to the beads.It was noted that recombinant LMO2 tended to bind nonspecifically, and that scFv was used for pull-down along with avidin agarose (see lanes 1 & 2, Figure 5F, G). This is likely due to partial denaturation of the protein during PAL incubation, which may explain the apparent partial inability of Abd-L9 to compete with PAL compounds (Figure 5F, lane 4 vs. lane 3).

[0233] LMO2 Abd compound activity in cells The specificity and titer of Abd-L compounds in cells were tested using dose-response BRET assays for different LMO2 PPIs. This included unmutated iDAb and iDAb dm3 This included LMO2 interactions with MAX, its natural partner proteins LDB1 and TAL1 (with E47) (Wadman et al., 1997), and unrelated control PPIs such as the bHLH interaction between MAX and CMYC. To develop various BRET assays, we first tested direct LMO2 interaction with TAL1 using a BRET donor saturation assay (Figure 14A), but this interaction was weak and did not result in high BRET 50 Values ​​were obtained. By individually adding partner proteins involved in the LMO2 complex (Wadman et al., 1997), it was found that simultaneous expression of heterodimerizing partners of E47 and TAL1 increased the relative affinity of LMO2-TAL1, resulting in the strongest binding affinity between LMO2 and TAL1 (Figure 14A, BRET). 50See the decrease in value (from 12.6 to 1). Unrelated interactions also occurred between BRET and LMO2-LDB1 (Figure 14B), as well as between CMYC bHLH and MAX bHLH (Figure 14C). Finally, the specificity of these three interactions was tested using a BRET competitive assay by co-expressing an untagged version of iDAb Ctl or iDAb LMO2 in BRET assay cells. iDAb LMO2 inhibited the BRET signal from LMO2-TAL1+E47 (Figure 14D) and the signal from LMO2-LDB1 (Figure 14E), but did not inhibit the signal from the MAX-CMYC interaction (Figure 14F).

[0234] Anti-LMO2 Abd compounds were evaluated in BRET dose-response assays using various BRET assays (Figures 6A-D and 14G, H). Except for Abd-L22 (approximately 85%), all compounds were LMO2-iDAb LMO2. dm3 BRET was not inhibited by more than 40-50%. However, regardless of whether the compound contained imidazolidinone substituents (Abd-L9 and L10) or piperazine substituents (Abd-L16), Abd-L9, Abd-L10, and Abd-L16 interacted at approximately 1 μM. LMO2-iDAb LMO2 dm3 The best IC 50 It was found to possess (Figure 6A and Table 1). Other compounds tested showed IC values ​​slightly above 7 μM and close to 50 μM for Abd-L19. 50 The values ​​are shown (Figures 6A, D and Table 1). When this group of compounds was assayed with LMO2-LDB1 BRET, little effect was observed except for Abd-L10, which showed only slight inhibition (1.2 μM IC5). 50Along with Abd-L10 at a maximum concentration of 35%, (Table 1 and Figures 6B, D). With respect to LMO2-iDAb LMO2 (unmutated iDAb) (Figure 6C), LMO2-TAL1+E47 (Figure 14G), or MAX bHLH-CMYC bHLH (Figure 14H), testing of this group of Abd-L compounds in BRET assays failed to show inhibition, even at the highest concentrations of compounds used throughout the series of BRET inhibition assays, with the exception of Abd-L22, which inhibits the LMO2-iDAb LMO2 interaction only at high concentrations (above 25 μM, Figure 6C).

[0235] Intracellular antibodies as templates for drug discovery Intracellular antibody fragments interact with proteins at either an antigenic site or at the site where the natural partner protein is involved in PPI. This provides an opportunity to use intracellular antibodies that induce compounds that are substitutes for specific interacting residues with the intracellular antibody. When the intracellular antibody directly interferes with PPI rather than using the natural partner protein, cellular antibodies can be obtained with very high affinity binding, as demonstrated for the binding of selected compounds to the RAS protein (Quevedo et al., 2018), demonstrating that this so-called undruggable target is actually drug-detectable. In vitro methods using intracellular antibodies as a drug discovery tool utilize competitive SPR with a chip that holds the target protein together with the interacting antibody at a predetermined position (Quevedo et al., 2018). RAS-binding compounds have been successful because of the very high affinity of the anti-RAS antibody, which limits the loss of antibody-antigen interaction on the SPR chip. As described herein, a similar approach using anti-LMO2 iDAbs was performed, avoiding the problem of iDAb loss during library screening by linking LMO2 and iDAb with a mobile linker. In this way, LMO2-binding chemicals were identified. Cell-based characterization testing of one of these compounds revealed undesirable characteristics.

[0236] As an alternative, the affinity of the iDAb to its target is not limited in cell-based assays such as the BRET assay described herein. Furthermore, the ability to manipulate affinity to a robust binder is readily available in antibodies, as only the primary sequence is used to identify the CDR for mutagenesis in a process called intracellular antibody dematuration (Tanaka & Rabbitts, manuscript in preparation). This process does not require structural information, and iDAbs of targets with low binding affinity can be used directly in cell-based approaches, thereby making this a flexible approach. Moreover, cell-based screening is also a more versatile approach because it can be performed on LMO2s that have escaped recombinant expression, except for co-expression with proteins that are difficult to express, such as LDB1 LID (Ryan et al., 2006) or iDAb (Sewell et al., 2014). Finally, an intrinsic advantage of cell-based assays, where the signal is generated by the direct interaction between the iDAb and the target, is that the compound already possesses the characteristics of cell entry, and we hereby present the LMO2 Abd-L series of our compounds.

[0237] LMO2-binding compounds derived from cell-based BRET2 chemical library screening The cell-based intracellular single-domain antibody-guided small molecule selection method described herein enables the direct identification of compounds that bind to the same region of iDAb. This has been illustrated with the T-cell oncogenic protein LMO2, which encodes an 18kDa polypeptide containing two zinc-binding LIM domains (Chambers and Rabbitts, 2015). These domains are interfaces that bind to class II basic helix-loop-helix (bHLH) transcription factors such as TAL1 / E2A and GATA (Wadman et al., 1994). Furthermore, these two DNA-binding complexes are cross-linked by the scaffold protein LDB1, which binds to LMO2 at interfaces different from those of transcription factors (Wadman et al., 1997). Anti-LMO2 iDAbs are characterized as inhibiting the oncogenic function of LMO2 in vivo by preventing LDB1 interaction, thereby preventing LMO2-dependent tumor growth in transplantation assays mediated by the disruption of the LMO2-multimer complex (Tanaka et al., 2011). More specifically, anti-LMO2 intracellular antibodies function as indirect PPI inhibitors through a novel mechanism that alters the native structure of LMO2. iDAb LMO2 induces conformational changes between the two LIM domains of LMO2 that are incompatible with LDB1 interaction and transcription factors (Sewell et al., 2014). For the Abd-L compounds selected here, no significant modification of the conformation of the LMO2 protein was observed, as shown in BRET data using TAL1 / E47: iDAb LMO2 prevented the binding of LMO2 to these proteins (Figure 14D), while the Abd-L compounds did not (Figure 14G).

[0238] Using iDAb, a library of 10,000 compounds binding to LMO2 was screened in an LMO2-iDAb BRET2 cell-based interaction assay. Many initial hits were obtained, and one chemical series of Abd-L5 to Abd-L12 was identified as a precursor. Direct binding of compounds Abd-L9, Abd-L10, and Abd-L13 was confirmed using recombinant LMO2-ΔLID protein in waterLOGSY NMR (Figure 5A-C). SAR analysis was performed on this chemical series to determine whether the compounds could tolerate larger groups and linkers, which allowed for the use of PAL technology. The use of the benzophenone moiety was tested as a photoaffinity label, and analogues with this group on the right-handed or left-handed side were confirmed to be still active. However, the linker could not be positioned on the left-handed side because this resulted in a loss of activity. Therefore, the compound Abd-L26 was produced by having a benzophenone photoreactive site on piperazine and biotin linked to aniline at the para position via a small polyethylene glycol (PEG) chain (Figure 5D). Crosslinking of Abd-L26 to the LMO2 protein confirmed its binding to the LMO2 protein (Figures 5E-F), which was inhibited by the addition of Abd-L9. These data support the conclusion that the chemical series is an intracellular antibody surrogate that binds to LMO2 when the anti-LMO2 iDAb comes into contact with LMO2. Cell-based selection involves competition among compounds for the interaction between the dematurated iDAb and LMO2, and these compounds exhibit their μM interference IC50. 50 As expected, it does not affect the interaction between the unmutated iDAb and LMO2 (except for Abd-L22 at the highest concentration). Furthermore, the compound does not bind to the LMO2-LID fusion, whereas it does bind to the LMO2-ΔLID fusion (if the LID is cleaved in part of the region where iDAb binds), and its binding site on LMO2 is further defined as the iDAb interaction region.

[0239] <Synthesis> Several methods for the chemical synthesis of heterocyclic carboxamide compounds of this application are described herein. These and / or other known methods may be modified and / or adapted in various ways to facilitate the synthesis of further compounds within the scope of this application and claims. Such alternative methods and modifications should be understood to be within the spirit and scope of this application and claims. Accordingly, the methods, schemes and examples described below are intended for illustrative purposes and should not be construed as limiting the scope of this disclosure.

[0240] All solvents and reagents were used as supplied (analytical or HPLC grade) without prior purification. Water was purified using the Elix® UV-10 system. Thin-layer chromatography was performed on aluminum plates coated with 60 F254 silica. The plates were visualized using ultraviolet light (254 nm) or a 1% KMnO4 aqueous solution. Flash column chromatography was performed on Kieselgel 60M silica in a glass column. NMR spectra were recorded using a Bruker Avance spectrometer (AVII400, AVIII400, AVIIIHD600, or AVIII700) in the specified deuterated solvent. The field was locked using an external reference for the relevant deuteron resonance. Chemical shifts (-) were reported in parts per million (ppm) with reference to the solvent peak. 1 The H spectrum is reported to two decimal places. 13C spectra are reported to one decimal place, and coupling constants (J) are cited in Hz (reported to one decimal place). The multiplicity of each signal is indicated by s (singlet); br.s (broad singlet); d (doublet); t (triplet); q (quadruplet); dd (doublet of doublet); td (doublet of triplet); qt (triplet of quadruplet); or m (multitlet). Low-resolution mass spectra (LRMS) were recorded from MeOH solution using a gilent 6120 spectrometer. Accurate mass measurements were performed using either a Bruker MicroTOF internally calibrated with polyalanine, or a Micromass GCT instrument with a Scientific Glass instrument BPX5 column (15 m × 0.25 mm) using amyl acetate as the lock mass, or by the Mass Spectrometry Division of the Chemistry Research Laboratory, University of Oxford, UK; m / z values ​​are reported in Daltons.

[0241] Basic Procedure A: Synthesis of substituted imidazolidinones (n=1) and pyrimidinones (n=2) [ka] The required (requisite) cyclic urea (1.0 equivalent) was dissolved in THF (10 mL) and cooled to 0°C before gradually adding NaH (60% suspension in oil, 1.0 equivalent). After 30 minutes, the suspension was treated with the required substituted benzyl bromide / benzyl chloride (0.9 equivalents). The resulting mixture was stirred for 2 hours (monitored by LC-MS and TLC) and warmed to room temperature before adding NH4Cl (saturated aqueous solution, 20 mL) and siRNA (20 mL). The aqueous layer was extracted with siRNA (2 × 20 mL), and the combined organic phase was washed with water (20 mL) and brine (20 mL saturated aqueous solution of sodium chloride), dried (Na2SO4), filtered under vacuum, and concentrated (using a rotary evaporator attached to a membrane pump). The crude material was purified with silica gel (5% MeOH in CH2Cl2) to obtain the desired compound as a colorless oil that solidified upon standing.

[0242] Basic Procedure B: Synthesis of Substituted Piperazines [ka] Boc-piperazine (1.1 equivalents) was dissolved in MeCN (5 mL) before sequentially adding the required substituted benzyl bromide / benzyl chloride (1.0 equivalent) following K2CO3 (2.5 equivalents). The resulting mixture was stirred for 18 hours before adding H2O / brine (1:1, 20 mL) and ethyl acetate (20 mL). The aqueous layer was extracted with ethyl acetate (20 mL), and the combined organic phase was dried (Na2SO4), filtered under vacuum, and concentrated. The crude substance was purified with silica gel (10% ethyl acetate in pentane) to obtain the title compound as a colorless oil that solidified upon standing. The product was dissolved in CH2Cl2 (5 mL) before adding TFA (500 μL). The resulting solution was stirred at room temperature for 18 hours and concentrated under vacuum. The compound was used in the next step without further purification.

[0243] Basic Procedure C: Palladium coupling reaction of substituted cyclic ureas to chlorooxazole (X=O) and chlorothiazole (X=S) [ka] The required substituted cyclic urea (1.1 equivalents), Cs2CO3 (3.0 equivalents), optimal ester-substituted chloroheterocyclic compound (1.0 equivalent), and X-Phos (10% mol) were sequentially added to a microwave vial, followed by degassed 1,4-dioxane (2 mL). Before adding Pd(OAc)2 (5% mol), the suspension was degassed for 5 minutes; before sealing the container, it was degassed with nitrogen for another 5 minutes, and the suspension was heated at 95°C for 24 hours. The reaction was cooled, diluted with SiO2 (10 mL), and washed with brine / water (1:1, 10 mL). The organic phase was dried (Na2SO4), filtered under vacuum, and concentrated. The crude material was purified over silica gel to obtain the desired compound.

[0244] Basic Procedure D: Synthesis of Substitutive Piperazyl Heterocycles [ka] Before adding the required chloro-heterocyclic compound (1.0 equivalent), substituted piperazine (1.2 equivalents) was dissolved in 1,4-dioxane / N,N-diisopropylethylamine (4:1.8 mL). The solution was stirred at 60°C for 48 hours, cooled to room temperature, diluted with SiO2O (30 mL), and washed with H2O / brine (1:1.20 mL). The organic phase was dried (Na2SO4), filtered under vacuum, and concentrated. The crude material was purified over silica gel to obtain the title compound.

[0245] Basic Procedure E: Basic hydrolysis of the ester moiety, followed by the HATU amide coupling reaction. [ka] Before adding NaOH (1M aqueous solution) to a pH > 8, the ester (1.0 equivalent) was dissolved in THF / MeOH (4:1). The resulting reaction was stirred at room temperature for 16 hours, and then acidified with HCl (1M aqueous solution) to a pH < 5. The solution was concentrated under vacuum, and the resulting carboxylic acid was used in the next step without further purification. Before sequentially adding N,N-diisopropylethylamine (3.0 equivalents), the required amine (1.2 equivalents), and HATU (1.4 equivalents), the acid was dissolved in DMF (2 mL). The resulting solution was stirred for 18 hours, diluted with SiO2 (10 mL), and washed with brine / water (1:1, 3 × 50 mL). The organic phase was dried (Na2SO4), filtered under vacuum, and concentrated. The crude material was purified over silica gel to obtain the title compound.

[0246] <Experimental Data> Abd-L5:1-benzylimidazolidinedion-2-one(1) [ka] Following basic procedure A using 2-imidazolidinone (1.00 g, 11.6 mmol, 1.0 equivalent) and benzyl bromide (1.25 mL, 10.4 mmol, 0.9 equivalents), the title compound 1 was obtained as a colorless oil that solidified upon standing after purification on silica gel (5% MeOH in CH2Cl2) (858 mg, 42%). m / z LRMS(ESI + ):177(100%)[M+H] + .

[0247] Ethyl 2-(3-benzyl-2-oxoimidazolidine-1-yl)thiazole-5-carboxylate (2) [ka] Following basic procedure C using cyclic urea 1 (102 mg, 0.578 mmol, 1.1 equivalent) and ethyl 2-chlorothiazole-5-carboxylate (100 mg, 0.525 mmol, 1.0 equivalent), the title compound 2 was obtained as a yellow oil (125 mg, 72%) on silica gel (3% MeOH in CH2Cl2). m / z LRMS (ESI + ):332(100%)[M+H] +

[0248] 2-(3-benzyl-2-oxoimidazolidine-1-yl)-N-(2-(methylsulfonamide)phenyl)thiazole-5-carboxamide(3)(Abd-L5) [ka] Following the basic procedure E using ester 2 and N-(2-aminophenyl)methanesulfonamide, the product was purified twice on silica gel (5% MeOH in CH2Cl2), and the title product was obtained as a dark yellow oil that solidified upon standing (32 mg, 41%). m / z LRMS(ESI + ):472(100%)[M+H] + HRMS(ESI+):C 21 H 22 N5O4 32 S2[M+H] + Calculated value: 472.1113, Measured value: 472.1120.

[0249] Abd-L6: Ethyl 2-(3-benzyl-2-oxoimidazolidine-1-yl)oxazole-4-carboxylate (4) [ka] Following basic procedure C using cyclic urea 1 (110 mg, 0.629 mmol, 1.1 equivalent) and ethyl 2-chlorooxazole-4-carboxylate (100 mg, 0.571 mmol, 1.0 equivalent), the product was purified on silica gel (3% MeOH in CH2Cl2) to obtain the title compound 4 as a yellow oil (106 mg, 59%). m / z LRMS(ESI + ):316(100%)[M+H] + .

[0250] 2-(3-benzyl-2-oxoimidazolidine-1-yl)-N-(4-phenoxyphenyl)oxazole-4-carboxamide(5)(Abd-L6) [ka] Following the basic procedure E using ester 4 and 4-phenoxyaniline, the product was purified on silica gel (5% MeOH in CH2Cl2), and the title product was obtained as a dark yellow oil that solidified upon standing (27 mg, 52%). m / z LRMS(ESI + ):455(100%)[M+H] + .HRMS(ESI + ):C 26 H 23 N4O4[M+H] + Calculated value: 455.1719, measured value: 455.1720.

[0251] Abd-L7:1-(3-chlorobenzyl)imidazolidined-2-one(6) [ka] Following basic procedure A using 2-imidazolidinone (500 mg, 5.80 mmol, 1.0 equivalent) and 3-chlorobenzyl bromide (685 μL, 5.20 mmol, 0.9 equivalents), the title compound 6 was obtained as a colorless oil that solidified upon standing after purification on silica gel (3% MeOH in CH2Cl2) (463 mg, 38%). m / z LRMS(ESI + ):211(100%)[M+H] + .

[0252] Ethyl 2-(3-(3-chlorobenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxylate (7) [ka] Following basic procedure C using cyclic urea 6 (132 mg, 0.629 mmol, 1.1 equivalent) and ethyl 2-chlorooxazole-4-carboxylate (100 mg, 0.571 mmol, 1.0 equivalent), the title compound 7 was obtained as a yellow oil (117 mg, 54%) on silica gel (3% MeOH in CH2Cl2). m / z LRMS (ESI + ):350(100%)[M+H] + .

[0253] N-(4-(1H-pyrrole-1-yl)phenyl)-2-(3-(3-chlorobenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxamide(8)(Abd-L7) [ka] Following the basic procedure E using ester 7 and 4-pyrroleaniline, the product was purified on silica gel (7% MeOH in CH2Cl2), and the title product was obtained as a dark yellow oil that solidified upon standing (27 mg, 52%). m / z LRMS(ESI + ):462(100%)[M+H] + .HRMS(ESI +):C 24 H 21 35 ClN5O3[M+H] + Calculated value: 462.1333, Measured value: 462.1332.

[0254] Abd-L8: 1-(4-chlorobenzyl)imidazolidined-2-one(9) [ka] Following basic procedure A using 2-imidazolidinone (500 mg, 5.80 mmol, 1.0 equivalent) and 4-chlorobenzyl bromide (1.07 g, 5.20 mmol, 0.9 equivalents), the title compound was obtained as a colorless oil that solidified upon standing after purification on silica gel (5% MeOH in CH2Cl2) (463 mg, 38%). m / z LRMS (ESI + ):211(100%)[M+H] + .

[0255] Ethyl 2-(3-(4-chlorobenzyl)-2-oxoimidazolidine-1-yl)oxazole-5-carboxylate (10) [ka] Following basic procedure C using cyclic urea 9 (132 mg, 0.629 mmol, 1.1 equivalent) and ethyl 2-chlorooxazole-5-carboxylate (100 mg, 0.571 mmol, 1.0 equivalent), the title compound 10 was obtained as a yellow oil (122 mg, 61%) after purification on silica gel (3% MeOH in CH2Cl2). m / z LRMS(ESI + ):350(100%)[M+H] + .

[0256] 2-(3-(4-chlorobenzyl)-2-oxoimidazolidine-1-yl)-N-(3,4-dimethoxyphenyl)oxazole-5-carboxamide(11)(Abd-L8) [ka] Following the basic procedure E using ester 10 and 3,4-dimethoxyaniline, the product was purified on silica gel (5% MeOH in CH2Cl2), and the title product was obtained as a dark brown oil that solidified upon standing (22 mg, 54%). m / z LRMS (ESI + ):457(100%)[M+H] + .HRMS(ESI + ):C 22 H 22 35 ClN4O5[M+H] + Calculated value: 457.1279, measured value: 457.1282.

[0257] Abd-L9:1-(3-methoxybenzyl)imidazolidined-2-one(12) [ka] Following basic procedure A using 2-imidazolidinone (1.00 g, 11-6 mmol, 1.0 equivalent) and 3-methoxybenzyl bromide (1.47 mL, 10.5 mmol, 0.9 equivalents), the title product was obtained as a colorless oil that solidified upon standing after purification on silica gel (5% MeOH in CH2Cl2) (1.02 g, 47%). m / z LRMS(ESI + ):207(100%)[M+H] + .

[0258] Ethyl 2-(3-(3-methoxybenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxylate (13) [ka] Following basic procedure C using cyclic urea 12 (82 mg, 0.396 mmol, 1.1 equivalent) and ethyl 2-chlorooxazole-4-carboxylate (64 mg, 0.360 mmol, 1.0 equivalent), the title compound 13 was obtained as a yellow oil (88 mg, 71%) after purification on silica gel (3% MeOH in CH2Cl2). m / z LRMS(ESI + ):346(100%)[M+H] + .

[0259] N-(4-(benzyloxy)phenyl)-2-(3-(3-methoxybenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxamide(14)(Abd-L9) [ka] Following the basic procedure E using ester 13 and 4-(benzyloxy)aniline, the title product was obtained as a dark yellow oil that solidified upon standing after purification on silica gel (5% MeOH in CH2Cl2) (137 mg, 62%). m / z LRMS(ESI + ):499(100%)[M+H] + .HRMS(ESI + ):C 28 H 27 N4O5[M+H] + Calculated value: 499.1981, measured value: 499.1978.

[0260] Abd-L10:3-((2-oxoimidazolidine-1-yl)methyl)benzonitrile(15) [ka] Following basic procedure A using 2-imidazolidinone (500 mg, 5.80 mmol, 1.0 equivalent) and 3-cyanobromide benzyl (1.01 g, 5.20 mmol, 0.9 equivalents), the title compound was obtained as a colorless oil that solidified upon standing after purification on silica gel (5% MeOH in CH2Cl2) (463 mg, 41%). m / z LRMS(ESI + ):202(100%)[M+H] +

[0261] Ethyl 2-(3-(3-cyanobenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxylate (16) [ka] Following basic procedure C using cyclic urea 15 (126 mg, 0.629 mmol, 1.1 equivalent) and ethyl 2-chlorooxazole-4-carboxylate (100 mg, 0.571 mmol, 1.0 equivalent), the title compound 16 was obtained as a yellow oil (109 mg, 56%) on silica gel (3% MeOH in CH2Cl2). m / z LRMS (ESI + ):341(100%)[M+H] + .

[0262] N-(4-(1H-pyrrole-1-yl)phenyl)-2-(3-(3-cyanobenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxamide(17)(Abd-L10) [ka] Following the basic procedure E using ester 16 and 4-pyrroleaniline, the product was purified on silica gel (7% MeOH in CH2Cl2), and then allowed to stand to solidify, resulting in the title product as a dark yellow oil (27 mg, 52%). m / z LRMS(ESI + ):453(100%)[M+H] + .HRMS(ESI + ):C 25 H21 N6O3[M+H] + Calculated value: 453.1675, measured value: 453.1674.

[0263] Abd-L11:2-(3-(4-chlorobenzyl)-2-oxoimidazolidine-1-yl)-N-phenethyloxazole-5-carboxamide(18)(Abd-L11) [ka] Following the basic procedure E using ester 10 and 2-phenylethane-1-amine, the title product was obtained as a dark yellow oil that solidified upon standing after purification on silica gel (5% MeOH in CH2Cl2) (18 mg, 49%). m / z LRMS(ESI + ):425(100%)[M+H] + .HRMS(ESI + ):C 22 H 22 35 ClN4O3[M+H] + Calculated value: 425.1380, Measured value: 425.1382.

[0264] Abd-L12:1-(2-chlorobenzyl)imidazolidined-2-one(19) [ka] Following basic procedure E using 2-imidazolidinone (500 mg, 5.80 mmol, 1.0 equivalent) and 2-chlorobenzyl bromide (680 μL, 5.20 mmol, 0.9 equivalents), the title compound was obtained as a colorless oil that solidified upon standing after purification on silica gel (5% MeOH in CH2Cl2) (463 mg, 38%). m / z LRMS(ESI + ):211(100%)[M+H] + .

[0265] Ethyl 2-(3-(2-chlorobenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxylate (20) [ka] Following basic procedure C using cyclic urea 19 (132 mg, 0.629 mmol, 1.1 equivalent) and ethyl 2-chlorooxazole-4-carboxylate (100 mg, 0.571 mmol, 1.0 equivalent), the title compound 20 was obtained as a yellow oil (98 mg, 49%) after purification on silica gel (3% MeOH in CH2Cl2). m / z LRMS(ESI + ):350(100%)[M+H] + .

[0266] 2-(3-(2-chlorobenzyl)-2-oxoimidazolidine-1-yl)-N-(4-phenoxyphenyl)oxazole-4-carboxamide(21)(Abd-L12) [ka] Following the basic procedure E using ester 20 and 4-phenoxyaniline, the product was purified on silica gel (5% MeOH in CH2Cl2), and the title product was obtained as a dark yellow oil that solidified upon standing (32 mg, 61%). m / z LRMS(ESI + ):489(100%)[M+H] + .HRMS(ESI + ):C 26 H 22 35 ClN4O4[M+H] + Calculated value: 489.1330, Measured value: 489.1332.

[0267] Abd-L13:2-(3-benzyl-2-oxoimidazolidine-1-yl)-N-(4-phenoxybenzyl)oxazole-4-carboxamide(22)(Abd-L13) [ka] Following the basic procedure E using ester 4 and (4-phenoxyphenyl)methaneamine, the title product was obtained as a dark yellow oil that solidified upon standing after purification on silica gel (5% MeOH in CH2Cl2) (17 mg, 42%). m / z LRMS(ESI + ):469(100%)[M+H] + .HRMS(ESI + ):C 27 H 25 N4O4[M+H] + Calculated value: 469.1876, measured value: 469.1874.

[0268] Abd-L14:2-(3-(2-chlorobenzyl)-2-oxoimidazolidine-1-yl)-N-(4-phenoxybenzyl)oxazole-4-carboxamide(23)(Abd-L14) [ka] Following the basic procedure E using ester 20 and (4-phenoxyphenyl)methaneamine, the title product was obtained as a dark orange oil that solidified upon standing after purification on silica gel (5% MeOH in CH2Cl2) (21 mg, 53%). m / z LRMS(ESI + ):503(100%)[M+H] + .HRMS(ESI + ):C 27 H 24 35 ClN4O4[M+H] + Calculated value: 503.1486, Measured value: 503.1484.

[0269] Abd-L15:1-(3-methoxybenzyl)piperazine(24) [ka] Following basic procedure B using Boc-piperazine (1.00 g, 53-6 mmol, 1.1 equivalent), K2CO3 (1.70 g, 12.2 mmol, 2.5 equivalent), and 3-methoxybenzyl bromide (683 μL, 4.88 mmol, 1.0 equivalent), the title compound was obtained as a dark, colorless oil that solidified upon standing after purification on silica gel (10% butyl in pentane). m / z LRMS(ESI + ):307(100%)[M+H] + .

[0270] Before adding TFA (500 μL), the product was dissolved in CH2Cl2 (5 mL). The resulting solution was stirred at room temperature for 18 hours and then concentrated under vacuum. The title compound was used in the next step without further purification (987 mg, 98% across both steps). m / z LRMS(ESI + ):207(100%)[M+H] + .

[0271] Ethyl 2-(4-(3-methoxybenzyl)piperazine-1-yl)oxazole-4-carboxylate (25) [ka] Following basic procedure D using piperazine 24 (150 mg, 0.728 mmol, 1.2 equivalents) and ethyl 2-chlorooxazole-4-carboxylate (116 mg, 0.661 mmol, 1.0 equivalent), the title compound 25 was obtained as a yellow oil (163 mg, 71%) after purification on silica gel (4% MeOH in CH2Cl2). m / z LRMS(ESI + ):346(100%)[M+H] + .

[0272] N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(3-methoxybenzyl)piperazine-1-yl)oxazole-4-carboxamide(26)(Abd-L15) [ka] Following the basic procedure E using ester 25 and 4-pyrroleaniline, the product was purified on silica gel (7% MeOH in CH2Cl2) to obtain the title product as a light brown powder (52 mg, 74%). m / z LRMS(ESI + ):458(100%)[M+H] + .HRMS(ESI + ):C 26 H 28 N5O3[M+H] + Calculated value: 458.2192, Measured value: 458.2192.

[0273] N-(4-(benzyloxy)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)oxazole-4-carboxamide(27)(Abd-L16) [ka] Following the basic procedure E using ester 26 and 4-(benzyloxy)aniline, the title product was obtained as a yellow oil that solidified upon standing after purification on silica gel (5% MeOH in CH2Cl2) (37 mg, 69%). m / z LRMS(ESI + ):499(100%)[M+H] + .HRMS(ESI + ):C 29 H 31 N4O4[M+H] + Calculated value: 499.2345, Measured value: 499.2345.

[0274] Abd-L17 Ethyl 2-(4-(3-methoxybenzyl)piperazine-1-yl)thiazole-4-carboxylate (28) [ka] Using piperazine 24 (150 mg, 0.728 mmol, 1.2 equivalents) and ethyl 2-chlorothiazole-4-carboxylate (126 mg, 0.661 mmol, 1.0 equivalent), the following basic procedure D was followed by purification on silica gel (4% MeOH in CH2Cl2) to obtain the title compound 28 as a yellow oil (212 mg, 89%). m / z LRMS(ESI + ):362(100%)[M+H] + .

[0275] N-(4-(benzyloxy)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)thiazole-4-carboxamide(29)(Abd-L17) [ka] Using ester 28 and 4-(benzyloxy)aniline, the product was purified on silica gel (4% MeOH in CH2Cl2) according to basic procedure E, and the title product was obtained as a yellow oil that solidified upon standing (42 mg, 78%). m / z LRMS(ESI + ):515(100%)[M+H] + .HRMS(ESI + ):C 29 H 31 N4O3 32 S[M+H] + Calculated value: 515.2117, measured value: 515.2116.

[0276] Abd-L18:N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(3-methoxybenzyl)piperazine-1-yl)thiazole-4-carboxamide(30)(Abd-L18) [ka] After purification using ester 28 and 4-pyrroleaniline according to basic procedure E on silica gel (4% MeOH in CH2Cl2), the title product was obtained as a beige solid (37 mg, 75%). m / z LRMS(ESI+ ):474(100%)[M+H] + .HRMS(ESI + ):C 29 H 31 N4O3 32 S[M+H] + Calculated value: 474.1964, measured value: 474.1965.

[0277] Abd-L19:1-(4-methoxybenzyl)piperazine(31) [ka] Using Boc-piperazine (1.00 g, 53-6 mmol, 1.1 equivalent), K2CO3 (1.70 g, 12.2 mmol, 2.5 equivalent), and 4-methoxybenzyl bromide (700 μL, 4.88 mmol, 1.0 equivalent), the title compound was obtained as a colorless oil that solidified upon standing after purification on silica gel (10% SiO in pentane) according to basic procedure B. m / z LRMS (ESI + ):307(100%)[M+H] + Before adding TFA (500 μL), the product was dissolved in CH2Cl2 (5 mL). The resulting solution was stirred at room temperature for 18 hours. The compound was concentrated under vacuum. Without further purification, the title compound was used in the next step (891 mg, 89% across two steps). m / z LRMS(ESI + ):207(100%)[M+H] + .

[0278] Ethyl 2-(4-(4-methoxybenzyl)piperazine-1-yl)oxazole-4-carboxylate (32) [ka] Using piperazine 31 (150 mg, 0.728 mmol, 1.2 equivalents) and ethyl 2-chlorooxazole-4-carboxylate (116 mg, 0.661 mmol, 1.0 equivalent), the title compound 32 was obtained as a yellow oil (163 mg, 71%) after purification on silica gel (4% MeOH in CH2Cl2) according to basic procedure D. m / z LRMS (ESI + ):346(100%)[M+H] + .

[0279] N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(4-methoxybenzyl)piperazin-1-yl)oxazole-4-carboxamide(33)(Abd-L19) [ka] After purification using ester 32 and 4-pyrroleaniline on silica gel (7% MeOH in CH2Cl2) according to basic procedure E, the title product was obtained as a brown powder (24 mg, 67%). m / z LRMS(ESI + ):458(100%)[M+H] + .HRMS(ESI + ):C 26 H 28 N5O3[M+H] + Calculated value: 458.2192, Measured value: 458.2192.

[0280] Abd-L201-(2-methoxybenzyl)piperazine(34) [ka] Using Boc-piperazine (200 mg, 1.07 mmol, 1.1 equivalent), K2CO3 (370 mg, 2.68 mmol, 2.5 equivalent), and 2-methoxybenzyl bromide (195 mg, 0.972 mmol, 1.0 equivalent), the title compound was obtained as a colorless oil that solidified upon standing after purification on silica gel (10% ¼ in pentane) according to basic procedure B. m / z LRMS(ESI +):307(100%)[M+H] + .

[0281] Before adding TFA (500 μL), the product was dissolved in CH2Cl2 (5 mL). The resulting solution was stirred at room temperature for 18 hours and then concentrated under vacuum. Without further purification, the title compound was used in the next step (164 mg, 82% across two steps). m / z LRMS(ESI + ):207(100%)[M+H] + .

[0282] Ethyl 2-(4-(2-methoxybenzyl)piperazine-1-yl)thiazole-4-carboxylate (35) [ka] Using piperazine 34 (100 mg, 0.728 mmol, 1.2 equivalents) and ethyl 2-chlorothiazole-4-carboxylate (126 mg, 0.661 mmol, 1.0 equivalent), the title compound 28 was obtained as a yellow oil after purification on silica gel (4% MeOH in CH2Cl2) according to basic procedure D. (216 mg, 92%) m / z LRMS (ESI + ):362(100%)[M+H] + .

[0283] N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(2-methoxybenzyl)piperazine-1-yl)thiazole-4-carboxamide(36)(Abd-L20) [ka] Using ester 35 and 4-pyrroleaniline, the product was purified on silica gel (6% MeOH in CH2Cl2) according to basic procedure E, and the title product was obtained as a yellow oil that solidified upon standing (28 mg, 72%). m / z LRMS(ESI + ):474(100%)[M+H] + .HRMS(ESI + ):C26 H 28 N5O3[M+H] + Calculated value: 474.1864, measured value: 474.1863.

[0284] 2-(4-(3-methoxybenzyl)piperazin-1-yl)-N-(4-(trifluoromethoxy)phenyl)thiazole-4-carboxamide(37)(Abd-L21) [ka] Using ester 28 and 4-(trifluoromethoxy)aniline, the product was purified on silica gel (4% MeOH in CH2Cl2) according to basic procedure E, and the title product was obtained as a dark yellow oil that solidified upon standing (28 mg, 76%). m / z LRMS(ESI + ):493(100%)[M+H] + .HRMS(ESI + ):C 23 H 24 F3N4O3 32 S[M+H] + Calculated value: 493.1521, Measured value: 493.1520.

[0285] 2-(4-(3-methoxybenzyl)piperazine-1-yl)-N-(6-methoxypyridine-3-yl)thiazole-4-carboxamide(38)(Abd-L22) [ka] After purification using ester 28 and 6-methoxypyridine-3-amine on silica gel (4% MeOH in CH2Cl2) according to basic procedure E, the title product was obtained as a red oil (23 mg, 71%). m / z LRMS(ESI + ):440(100%)[M+H] + .HRMS(ESI + ):C 22 H 25 N5O3 32 S[M+H] +Calculated value: 440.1756, measured value: 440.1754.

[0286] 2-(4-(3-methoxybenzyl)piperazine-1-yl)-N-(2-methoxypyrimidine-5-yl)thiazole-4-carboxamide(39)(Abd-L23) [ka] After purification using ester 28 and 6-methoxypyridine-3-amine on silica gel (4% MeOH in CH2Cl2) according to basic procedure E, the title product was obtained as a pale yellow oil (23 mg, 71%). m / z LRMS(ESI + ):441(100%)[M+H] + .HRMS(ESI + ):C 21 H 25 N6O3 32 S[M+H] + Calculated value: 441.1709, measured value: 441.1710.

[0287] Abd-L24:1-(3-methoxybenzyl)tetrahydropyrimidine-2(1H)-one(40) [ka] Using tetrahydro-2(1H)-pyrimidinone (500 mg, 5.00 mmol, 1.0 equivalent), NaH (60% suspension in oil, 134 mg, 5.00 mmol, 1.0 equivalent), and 3-methoxybenzyl bromide (630 μL, 4.50 mmol, 0.9 equivalents), the oil was purified on silica gel (5% MeOH in CH2Cl2) according to basic procedure A, and a colorless oil-based title compound that solidified upon standing was obtained (564 mg, 57%). m / z LRMS(ESI + ):221(100%)[M+H] + .

[0288] Ethyl 2-(3-(3-methoxybenzyl)-2-oxotetrahydropyrimidine-1(2H)-yl)oxazole-4-carboxylate (41) [ka] Cyclic urea 40 (132 mg, 0.375 mmol, 1.1 equivalent) and ethyl 2-chlorooxazole-4-carboxylate (60 mg, 0.341 mmol, 1.0 equivalent) were purified on silica gel (3% MeOH in CH2Cl2) according to basic procedure C, and the title compound 41 was obtained as a yellow oil (51 mg, 42%). m / z LRMS(ESI + ):360(100%)[M+H] + .

[0289] N-(4-(1H-pyrrole-1-yl)phenyl)-2-(3-(3-methoxybenzyl)-2-oxotetrahydropyrimidine-1(2H)-yl)oxazole-4-carboxamide(42)(Abd-L24) [ka] After purifying twice on silica gel (5% MeOH in CH2Cl2) using ester 41 and 4-pyrroleaniline according to basic procedure E, the title product was obtained as a dark yellow oil (12 mg, 41%). m / z LRMS(ESI + ):472(100%)[M+H] + .HRMS(ESI + ):C 26 H 26 N5O4[M+H] + Calculated value: 472.1985, measured value: 472.1986.

[0290] Abd-L25: Ethyl 2-(4-(3-methoxybenzyl)piperazin-1-yl)pyrimidine-4-carboxylate (43) [ka] Using piperazine 24 (133 mg, 0.645 mmol, 1.2 equivalents) and ethyl 2-chloropyrimidine-4-carboxylate (100 mg, 0.538 mmol, 1.0 equivalent), the compound was purified on silica gel (4% MeOH in CH2Cl2) according to basic procedure D, and the title compound 43 was obtained as a pale yellow oil (178 mg, 93%). m / z LRMS(ESI + ):357(100%)[M+H] + .

[0291] N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(3-methoxybenzyl)piperazine-1-yl)pyrimidine-4-carboxamide(44)(Abd-L25) [ka] After purification using ester 43 and 4-pyrroleaniline according to basic procedure E on silica gel (4% MeOH in CH2Cl2), the title product was obtained as a beige solid (35 mg, 89%). m / z LRMS(ESI + ):469(100%)[M+H] + .HRMS(ESI + ):C 27 H 29 N6O2[M+H] + Calculated value: 469.2352, Measured value: 469.2353.

[0292] Abd-L26 (PAL compound): Phenyl(4-(piperazine-1-ylmethyl)phenyl)methanone (45) [ka] Using Boc-piperazine (1.00 g, 53-6 mmol, 1.1 equivalent), K2CO3 (1.70 g, 12.2 mmol, 2.5 equivalent), and 4-bromomethylbenzophenone (1.34 g, 4.88 mmol, 1.0 equivalent), a colorless oil-based compound was obtained by purification on silica gel (8% SiO in pentane) according to basic procedure B, followed by standing to solidify. m / z LRMS (ESI + ):381(100%)[M+H] + Before adding TFA (1 mL), the product was dissolved in CH2Cl2 (10 mL). The resulting solution was stirred at room temperature for 18 hours and then concentrated under vacuum. Without further purification, the title compound was used in the next step (1.32 g, 88% across two steps). m / z LRMS(ESI + ):281(100%)[M+H] + .

[0293] Ethyl 2-(4-(4-benzylbenzyl)piperazine-1-yl)oxazole-4-carboxylate (46) [ka] Using piperazine 45 (580 mg, 2.07 mmol, 1.2 equivalents) and ethyl 2-chlorooxazole-4-carboxylate (326 mg, 1.86 mmol, 1.0 equivalent), the following basic procedure D was followed by purification on silica gel (3% MeOH in CH2Cl2) to obtain the title compound 46 as a yellow oil (630 mg, 81%). m / z LRMS(ESI + ):420(100%)[M+H] + .

[0294] N-(4-aminophenyl)-2-(4-(4-benzylbenzyl)piperazine-1-yl)oxazole-4-carboxamide(47) [ka] Using ester 46 and t-butyl-4-aminophenylcarbamate, the Boc protected product was obtained (82 mg, 73%) as a pale yellow oil that solidified upon standing after purification on silica gel (25% Â in CH2Cl2) according to basic procedure E. m / z LRMS (ESI + ):582(100%)[M+H] + Before adding TFA (150 μL), the product was dissolved in CH2Cl2 (2 mL). The resulting solution was stirred at room temperature for 18 hours and concentrated under vacuum. The title compound was used in the next step without further purification (66 mg, 99%). m / z LRMS(ESI + ):482(100%)[M+H] + .

[0295] Benzyl(2-(2-(2-((4-(2-(4-(4-benzylbenzyl)piperazin-1-yl)oxazole-4-carboxamide)phenyl)amino)-2-oxoethoxy)ethoxy)ethyl)carbamate(48) [ka] Before sequentially adding N,N-diisopropylethylamine (65 μL, 0.375 mmol, 3.0 equivalents), 3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecane-12-euic acid (45 mg, 0.150 mmol, 1.2 equivalents), and HATU (67 mg, 0.175 mmol, 1.4 equivalents), aniline 47 (60 mg, 0.125 mmol, 1.0 equivalent) was dissolved in DMF (2 mL). The resulting solution was stirred for 18 hours, diluted with SiO2 (10 mL), and washed with brine / water (1:1, 3 × 50 mL). The organic phase was dried (Na2SO4), filtered under vacuum, and concentrated. The crude material was then purified on silica gel (CH2Cl2 with 9% MeOH) to obtain the title compound as a yellow oil (72 mg, 76%). m / z LRMS(ESI + ):761(100%)[M+H] + .

[0296] 2-(4-(4-benzylbenzyl)piperazine-1-yl)-N-(4-(2-(2-(2-(5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazole-4-yl)pentanamide)ethoxy)ethoxy)acetamide)phenyl)oxazole-4-carboxamide(49)(Abd-L26) [ka] Carbamate 48 was dissolved in THF (2 mL) before adding MeOH (200 μL). The solution was degassed with nitrogen for 5 minutes before adding a catalytic amount of Pd / C. The suspension was degassed with nitrogen for another 5 minutes before exchanging the atmosphere with hydrogen (by balloon). The reaction was monitored by TLC and MS, and after 2 hours, the reaction was complete. The balloon was removed, and the reaction was passed through a Celite pad using SiO and MeOH as eluents. The product was used in the next step without further purification. The resulting amine (32 mg, 0.050 mmol, 1.0 equivalent) was dissolved in DMF (1 mL) before sequentially adding N,N-diisopropylethylamine (26 μL, 0.150 mmol, 3.0 equivalents), D-biotin (15 mg, 0.060 mmol, 1.2 equivalents), and HATU (27 mg, 0.070 mmol, 1.4 equivalents). The resulting solution was stirred for 18 hours, diluted with dimethylammonium chloride (10 mL), and washed with brine / water (1:1, 3 × 20 mL). The organic phase was dried (Na₂SO₄), filtered under vacuum, and concentrated. The crude substance was purified on silica gel (10% MeOH in CH₂Cl₂) and further purified by preparative TLC (15% MeOH in CH₂Cl₂) to obtain the title compound (12 mg, 28%) as a yellow oil. m / z LRMS(ESI + ):853(100%)[M+H] + .

[0297] Specific embodiments of the present invention are described herein for reference and illustrative purposes, but it will be apparent to those skilled in the art that various modifications can be made without departing from the scope of the invention as defined by the appended claims.

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Cochet, O., Kenigsberg, M., Delumeau, I., Virone-Oddos, A., Multon, M.C., Fridman, W.H., Schweighoffer, F., Teillaud, J.L., and Tocque, B. (1998). Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression. Cancer Res 58, 1170-1176. Cruz-Migoni, A., Canning, P., Quevedo, C.E., Bataille, C.J.R., Bery, N., Miller, A., Russell, A.J., Phillips, S.E.V., Carr, S.B., and Rabbitts, T.H. (2019). Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds. Proc Natl Acad Sci U S A 116, 2545-2550. Dalvit, C., Fogliatto, G., Stewart, A., Veronesi, M., and Stockman, B. (2001). WaterLOGSY as a method for primary NMR screening: practical aspects and range of applicability. J Biomol NMR 21, 349-359. Ferrando, A.A., and Look, A.T. (2003). Gene expression profiling in T-cell acute lymphoblastic leukemia. Semin Hematol 40, 274-280. Gupta, A., Xu, J., Lee, S., Tsai, S.T., Zhou, B., Kurosawa, K., Werner, M.S., Koide, A., Ruthenburg, A.J., Dou, Y., et al. (2018). Facile target validation in an animal model with intracellularly expressed monobodies. Nat Chem Biol 14, 895-900. Lavoie, H., Thevakumaran, N., Gavory, G., Li, J.J., Padeganeh, A., Guiral, S., Duchaine, J., Mao, D.Y., Bouvier, M., Sicheri, F., et al. (2013). Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization. Nat Chem Biol 9, 428-436. McCormack, M.P., Forster, A., Drynan, L., Pannell, R., and Rabbitts, T.H. (2003). The LMO2 T-cell oncogene is activated via chromosomal translocations or retroviral insertion during gene therapy but has no mandatory role in normal T-cell development. Mol Cell Biol 23, 9003-9013. Nam, C.H., Lobato, M.N., Appert, A., Drynan, L.F., Tanaka, T., and Rabbitts, T.H. (2008). An antibody inhibitor of the LMO2-protein complex blocks its normal and tumorigenic functions. Oncogene 27, 4962-4968. Quevedo, C.E., Cruz-Migoni, A., Bery, N., Miller, A., Tanaka, T., Petch, D., Bataille, C.J.R., Lee, L.Y.W., Fallon, P.S., Tulmin, H., et al. (2018). Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment. Nat Commun 9, 3169. Rabbitts, T.H. (2009). Commonality but diversity in cancer gene fusions. Cell 137, 391-395. Royer-Pokora, B., Loos, U., and Ludwig, W.D. (1991). TTG-2, a new gene encoding a cysteine-rich protein with the LIM motif, is overexpressed in acute T-cell leukaemia with the t(11;14)(p13;q11). Oncogene 6, 1887-1893. Ryan, D.P., Sunde, M., Kwan, A.H., Marianayagam, N.J., Nancarrow, A.L., Vanden Hoven, R.N., Thompson, L.S., Baca, M., Mackay, J.P., Visvader, J.E., et al. (2006). Identification of the key LMO2-binding determinants on Ldb1. J Mol Biol 359, 66-75. Sadakane, Y., and Hatanaka, Y. (2006). Photochemical fishing approaches for identifying target proteins and elucidating the structure of a ligand-binding region using carbene-generating photoreactive probes. Anal Sci 22, 209-218. Scott, D.E., Bayly, A.R., Abell, C., and Skidmore, J. (2016). Small molecules, big targets: drug discovery faces the protein-protein interaction challenge. Nat Rev Drug Discov 15, 533-550. Sewell, H., Tanaka, T., El Omari, K., Mancini, E.J., Cruz, A., Fernandez-Fuentes, N., Chambers, J., and Rabbitts, T.H. (2014). Conformational flexibility of the oncogenic protein LMO2 primes the formation of the multi-protein transcription complex. Sci Rep 4, 3643. Smith, E., and Collins, I. (2015). Photoaffinity labeling in target- and binding-site identification. Future Med Chem 7, 159-183. Spencer-Smith, R., Koide, A., Zhou, Y., Eguchi, R.R., Sha, F., Gajwani, P., Santana, D., Gupta, A., Jacobs, M., Herrero-Garcia, E., et al. (2017). Inhibition of RAS function through targeting an allosteric regulatory site. Nat Chem Biol 13, 62-68. Tanaka, T., and Rabbitts, T.H. (2003). Intrabodies based on intracellular capture frameworks that bind the RAS protein with high affinity and impair oncogenic transformation. EMBO J 22, 1025-1035. Tanaka, T., and Rabbitts, T.H. (2008). Interfering with protein-protein interactions: potential for cancer therapy. Cell Cycle 7, 1569-1574. Tanaka, T., and Rabbitts, T.H. (2010). Interfering with RAS-effector protein interactions prevent RAS-dependent tumour initiation and causes stop-start control of cancer growth. Oncogene 29, 6064-6070. Tanaka, T., Sewell, H., Waters, S., Phillips, S.E., and Rabbitts, T.H. (2011). Single domain intracellular antibodies from diverse libraries: emphasizing dual functions of LMO2 protein interactions using a single VH domain. J Biol Chem 286, 3707-3716. Tanaka, T., Williams, R.L., and Rabbitts, T.H. (2007). Tumour prevention by a single antibody domain targeting the interaction of signal transduction proteins with RAS. EMBO J 26, 3250-3259. Visintin, M., Tse, E., Axelson, H., Rabbitts, T.H., and Cattaneo, A. (1999). Selection of antibodies for intracellular function using a two-hybrid in vivo system. Proc Natl Acad Sci U S A 96, 11723-11728. Wadman, I., Li, J., Bash, R.O., Forster, A., Osada, H., Rabbitts, T.H., and Baer, R. (1994). Specific in vivo association between the bHLH and LIM proteins implicated in human T cell leukemia. EMBO J 13, 4831-4839. Wadman, I.A., Osada, H., Grutz, G.G., Agulnick, A.D., Westphal, H., Forster, A., and Rabbitts, T.H. (1997). The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1 / NLI proteins. EMBO J 16, 3145-3157.

Claims

1. The following structural formula (I): 【Chemistry 1】 (In the formula, R １ teeth, (i) Formula: 【Chemistry 2】 (In the formula, 【Transformation 3】 This indicates the connection point; n is either 0 or 1; R １ａ and R １ｂ is selected from hydrogen or methyl; X ４ , X ５ and X ６ C-H, CR ａ The base of (or selected from N); (ii) Formula: 【Chemistry 4】 (In the formula, 【Transformation 5】 This indicates the connection point; n, R １ａ and R １ｂ are as defined above; Ring B is a saturated or partially unsaturated ring; X ７ , X ８ and X ９ In the case where the bond connecting adjacent atoms is an unsaturated double bond, C-H, C-R ａ Alternatively, if selected from N, or if the bond connecting them to an adjacent atom is a single bond, C-H ２ C-HR ａ , C-(R ａ ) ２ N-H, N-R ｂ The base of (selected from S or O); Selected from, Here, R ａ Each of these independently comprises (1-4C) alkyl, halo, (1-4C) haloalkyl, (1-4C) haloalkoxy, cyano, nitro, (3-6C) cycloalkyl, (3-6C) cycloalkyl(1-2C) alkyl, phenyl, (CH ２ ) ｑ１ NR ａｂ R ａｃ , (CH ２ ) ｑ１ OR ａｂ , (CH ２ ) ｑ１ C(O)R ａｂ , (CH ２ ) ｑ１ C(O)OR ａｂ , (CH ２ ) ｑ１ OC(O)R ａｂ , (CH ２ ) ｑ１ C(O)N(R) ａｃ ) R ａｂ , (CH ２ ) ｑ１ N(R) ａｃ ) C(O)R ａｂ , (CH ２ ) ｑ１ S(O) ｐ R ａｂ (p is 0, 1, or 2), (CH ２ ) ｑ１ SO ２ N(R) ａｃ ) R １ａｂ , or (CH ２ ) ｑ１ N(R) ａｃ ) SO ２ R ａｂ Selected from, Here, q1 is 0, 1, 2, or 3; R ａｂ This is selected from hydrogen, (1-4C)alkyl, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)alkyl, aryl, aryl(1-2C)alkyl, heteroaryl, heteroaryl(1-2C)alkyl, heterocyclyl, and heterocyclyl(1-2C)alkyl, and R ａｂ The following are optional: oxo, (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)aminoalkyl, (1-4C)hydroxyalkyl, cyano, nitro, NR ａｄ R ａｅ , OR ａｄ , C(O)R ａｄ , C(O)OR ａｄ OC(O)R ａｄ , C(O)N(R ａｅ ) R ａｄ , N(R ａｅ ) C(O)R ａｄ , S(O) ｐ R ａｄ (p is 0, 1, or 2), SO ２ N(R) ａｅ ) R ａｄ , N(R ａｅ ) SO ２ R ａｄ , or (CH ２ ) ｑ２ NR ａｄ R ａｅ Further substituted by one or more substituents independently selected from (q2 is 1, 2, or 3); where R ａｄ and R ａｅ Each is independently selected from hydrogen or (1-6C) alkyl; and R ａｃ is selected from hydrogen or (1-2C) alkyl; or R ａｂ and R ａｃ When they are bonded to a common N atom, each of them, along with the N atom to which they are bonded, is R as described above. ａｂ They can be linked together to form a 5 or 6-membered heteroaryl ring or a 5- to 7-membered heterocyclic ring that can be optionally substituted in the same manner; R ｂ However, (1-4C) alkyl, (1-4C) haloalkyl or -C(O)R ｂａ Selected independently from, R ｂａ is selected from (1-4C)alkyl, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)alkyl, aryl, aryl(1-2C)alkyl, heteroaryl, heteroaryl(1-2C)alkyl, heterocyclyl and heterocyclyl(1-2C)alkyl, and R ｂａ is oxo, (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)aminoalkyl, (1-4C)hydroxyalkyl, cyano, nitro, NR ｂｄ R ｂｅ , OR ｂｄ , C(O)R ｂｄ , C(O)OR ｂｄ , OC(O)R ｂｄ , C(O)N(R ｂｅ )R ｂｄ , N(R ｂｅ )C(O)R ｂｄ , S(O) ｐ R ｂｄ (p is 0, 1 or 2), SO ２ N(R ｂｅ )R ｂｄ , N(R ｂｅ )SO ２ R ｂｄ , or (CH ２ ) ｑ３ NR ｂｄ R ｂｅ (q3 is 1, 2 or 3) and is optionally further substituted by one or more substituents independently selected therefrom; R ｂｄ and R ｂｅ are each independently selected from hydrogen or (1-6C)alkyl; X １ N is X ２ is O or S, and X ３ This is CR6, and here, R ６ is hydrogen; Q is the formula: 【Transformation 6】 And; R ２ and R ３ is hydrogen; R ４ (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)aminoalkyl, (1-4C)hydroxyalkyl, cyano, nitro, NR ４ａ R ４ｂ , OR ４ａ , C(O)R ４ａ , C(O)OR ４ａ OC(O)R ４ａ , C(O)N(R ４ｂ ) R ４ａ , N(R ４ｂ ) C(O)R ４ａ , S(O) ｐ R ４ａ (p is 0, 1, or 2), SO ２ N(R) ４ｂ ) R ４ａ , N(R ４ｂ ) SO ２ R ４ａ , or (CH ２ ) ｑ４ NR ４ａ R ４ｂ A phenyl, heteroaryl, or heterocyclyl ring optionally substituted by (q4 is 1, 2, or 3): where R ４ａ This is selected from hydrogen, (1-4C) alkyl, (3-6C) cycloalkyl, (3-6C) cycloalkyl(1-2C) alkyl, phenylaryl(1-2C) alkyl, heteroaryl, heteroaryl(1-2C) alkyl, heterocyclyl, and heterocyclyl(1-2C) alkyl, and R ４ａ The following are optionally selected: (1-4C) alkyl, halo, (1-4C) haloalkyl, (1-4C) haloalkoxy, cyano, nitro, NR ４ａａ R ４ａｂ , OR ４ａａ , C(O)R ４ａａ , C(O)OR ４ａａ OC(O)R ４ａａ , C(O)N(R ４ａｂ ) R ４ａａ , N(R ４ａｂ ) C(O)R ４ａａ , S(O) ｐ R ４ａａ (p is 0, 1, or 2), SO ２ N(R) ４ａｂ ) R ４ａａ , N(R ４ａｂ ) SO ２ R ４ａａ , or (CH ２ ) ｑ５ NR ４ａａ R ４ａｂ (q5 is 1, 2, or 3) is further substituted by R ４ａａ and R ４ａｂ is hydrogen or (1-2C) alkyl; R ４ｂ is selected from hydrogen or (1-2C) alkyl; or R ４ａ and R ４ｂ When they are bonded to a common N atom, each of them, along with the N atom to which they are bonded, is R as described above. ４ They can be linked together to form a 5 or 6-membered heteroaryl ring or a 5- to 7-membered heterocyclic ring that can be optionally substituted in the same manner. A compound having, or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof.

2. R １ but, (i) Formula: 【Transformation 7】 (In the formula, 【Transformation 8】 This indicates the connection point; n is either 0 or 1; R １ａ and R １ｂ It is selected from hydrogen; X ４ , X ５ and X ６ C-H, CR ａ The base of (or selected from N); (ii) Formula: 【Chemistry 9】 (In the formula, 【Chemistry 10】 This indicates the connection point; n, R １ａ and R １ｂ This is defined as above; Ring B is a saturated ring; X ７ , X ８ and X ９ (Selected from C-H2) Selected from, Here, R ａ Each of these independently consists of methyl, halo, and (CH ２ ) ｑ１ NR ａｂ R ａｃ , (CH ２ ) ｑ１ OR ａｂ , (CH ２ ) ｑ１ C(O)R ａｂ or (CH ２ ) ｑ１ C(O)OR ａｂ Selected from, and q1 is 0; R ａｂ is selected from hydrogen, (1-4C)alkyl, aryl, and aryl(1-2C)alkyl, and R ａｂ is optionally further substituted by one or more substituents independently selected from the halo; and R ａｃ is selected from hydrogen; or R ａｂ and R ａｃ When they are bonded to a common N atom, each of them, along with the N atom to which they are bonded, is R as described above. ａｂ The compound according to claim 1, or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof, wherein they can be linked together to form a five-membered heteroaryl ring or a five- or six-membered heterocyclic ring that is optionally substituted in the same manner.

3. R １ but, (i) Formula: 【Chemistry 11】 (In the formula, 【Chemistry 12】 This indicates the connection point; n is either 0 or 1; R １ａ and R １ｂ It is selected from hydrogen; X ４ , X ５ and X ６ C-H, CR ａ The base of (or selected from N); (ii) Formula: 【Chemistry 13】 (In the formula, 【Chemistry 14】 This indicates the connection point; n is 0; R １ａ and R １ｂ It is selected from hydrogen; Ring B is a saturated ring; X ７ , X ８ and X ９ (Selected from C-H2) Selected from, Here, R ａ Each of these independently consists of methyl, halo, and (CH ２ ) ｑ１ NR ａｂ R ａｃ , (CH ２ ) ｑ１ OR ａｂ , (CH ２ ) ｑ１ C(O)R ａｂ or (CH ２ ) ｑ１ C(O)OR ａｂ Selected from, and q1 is 0; R ａｂ is selected from hydrogen, (1-2C) alkyl, phenyl, and benzyl, and R ａｂ is optionally further substituted by one or more substituents independently selected from the halo; and R ａｃ is selected from hydrogen; or R ａｂ and R ａｃ When they are bonded to a common N atom, each of them, along with the N atom to which they are bonded, is R as described above. ａｂ A compound according to claim 1 or 2, or a pharmaceutically acceptable salt, aqueous compound or solvated compound thereof, which can be linked together to form a five-membered heteroaryl ring or a five- or six-membered heterocyclic ring that is optionally substituted in the same manner.

4. R １ but, 【Chemistry 15】 Selected from, 【Chemistry 16】 A compound according to any one of claims 1 to 3, wherein indicates a bonding site, or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof.

5. R ４ (1-2C)alkyl, halo, (1-2C)haloalkyl, (1-2C)haloalkoxy, (1-2C)aminoalkyl, (1-2C)hydroxyalkyl, cyano, nitro, NR ４ａ R ４ｂ , OR ４ａ , C(O)R ４ａ , C(O)OR ４ａ OC(O)R ４ａ , C(O)N(R ４ｂ ) R ４ａ , N(R ４ｂ ) C(O)R ４ａ , S(O) ｐ R ４ａ (p is 0, 1, or 2), SO ２ N(R) ４ｂ ) R ４ａ , N(R ４ｂ ) SO ２ R ４ａ , or (CH ２ ) ｑ４ NR ４ａ R ４ｂ A phenyl, heteroaryl, or heterocyclyl ring optionally substituted by (q4 is 1, 2, or 3); where R ４ａ This is selected from hydrogen, (1-2C) alkyl, phenylaryl(1-2C)alkyl, heteroaryl, heteroaryl(1-2C)alkyl, heterocyclyl, and heterocyclyl(1-2C)alkyl; R ４ａ It is optionally further substituted with (1-2C)alkyl, halo, (1-2C)haloalkyl, (1-2C)haloalkoxy, cyano, or nitro; R ４ｂ is selected from hydrogen or (1-2C) alkyl; or R ４ａ and R ４ｂ They are linked to a common N atom, and together with the N atom to which they are linked, each of them is the R mentioned above. ４ A compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, aqueous compound or solvated compound thereof, which can be linked together to form a 5 or 6-membered heteroaryl ring or a 5- to 7-membered heterocyclic ring that is optionally substituted in the same manner.

6. R ４ However, halo, cyano, nitro, NR ４ａ R ４ｂ , OR ４ａ , C(O)R ４ａ , N(R ４ｂ ) C(O)R ４ａ A phenyl ring that is optionally substituted by; where R ４ａ is selected from hydrogen, (1-2C) alkyl, and phenylaryl(1-2C)alkyl; and R ４ｂ The compound according to any one of claims 1 to 5, wherein is selected from hydrogen, or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof.

7. R ４ but, 【Chemistry 20】 Selected from, 【Chemistry 21】 A compound according to any one of claims 1 to 6, wherein indicates a bonding site, or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof.

8. below: 2-(3-benzyl-2-oxoimidazolidine-1-yl)-N-(4-phenoxyphenyl)oxazole-4-carboxamide (Abd-L6); N-(4-(1H-pyrrole-1-yl)phenyl)-2-(3-(3-chlorobenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxamide (Abd-L7); N-(4-(benzyloxy)phenyl)-2-(3-(3-methoxybenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxamide (Abd-L9); N-(4-(1H-pyrrole-1-yl)phenyl)-2-(3-(3-cyanobenzyl)-2-oxoimidazolidine-1-yl)oxazole-4-carboxamide (Abd-L10); 2-(3-(2-chlorobenzyl)-2-oxoimidazolidine-1-yl)-N-(4-phenoxyphenyl)oxazole-4-carboxamide(Abd-L12); 2-(3-benzyl-2-oxoimidazolidine-1-yl)-N-(4-phenoxybenzyl)oxazole-4-carboxamide (Abd-L13); 2-(3-(2-chlorobenzyl)-2-oxoimidazolidine-1-yl)-N-(4-phenoxybenzyl)oxazole-4-carboxamide (Abd-L14); N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(3-methoxybenzyl)piperazine-1-yl)oxazole-4-carboxamide (Abd-L15); N-(4-(benzyloxy)phenyl)-2-(4-(3-methoxybenzyl)piperazine-1-yl)oxazole-4-carboxamide (Abd-L16); N-(4-(benzyloxy)phenyl)-2-(4-(3-methoxybenzyl)piperazine-1-yl)thiazole-4-carboxamide (Abd-L17); N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(3-methoxybenzyl)piperazine-1-yl)thiazole-4-carboxamide (Abd-L18); N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(4-methoxybenzyl)piperazine-1-yl)oxazole-4-carboxamide (Abd-L19); N-(4-(1H-pyrrole-1-yl)phenyl)-2-(4-(2-methoxybenzyl)piperazine-1-yl)thiazole-4-carboxamide (Abd-L20); 2-(4-(3-methoxybenzyl)piperazine-1-yl)-N-(4-(trifluoromethoxy)phenyl)thiazole-4-carboxamide (Abd-L21); 2-(4-(3-methoxybenzyl)piperazine-1-yl)-N-(6-methoxypyridine-3-yl)thiazole-4-carboxamide (Abd-L22); and 2-(4-(3-methoxybenzyl)piperazine-1-yl)-N-(2-methoxypyrimidine-5-yl)thiazole-4-carboxamide (Abd-L23); A compound, or a pharmaceutically acceptable salt thereof, a aqueous compound, or a solvated compound, selected from any one of the following.

9. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, aqueous compound, or solvated compound thereof, in a mixture with a pharmaceutically acceptable diluent or carrier.

10. A pharmaceutical composition according to claim 9, used in therapeutic purposes.

11. The pharmaceutical composition according to claim 9, which is used to inhibit cell proliferation, such as in the treatment of cancer.

12. The pharmaceutical composition according to claim 11, wherein the cancer is a blood cancer.

13. Use of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, aqueous compound or solvated compound thereof, or the pharmaceutical composition according to claim 9, in the manufacture of a pharmaceutical for treating a proliferative disorder in a patient requiring such treatment.

14. The use of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, aqueous compound or solvated compound thereof, or a pharmaceutical composition according to claim 9, in the manufacture of a pharmaceutical for treating cancer in patients requiring such treatment.

15. The use according to claim 14, wherein the cancer is a blood cancer.

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