Benzimidazole derivatives as kinase inhibitors

Abstract

The present invention relates to derivatives of benzimidazoles of formula (I) as disclosed herein as well as pharmaceutical compositions comprising said derivatives. The derivates according to the present invention are serine/threonine and tyrosine kinase-inhibitors, particularly of PIM1-3- and DYRK1A-kinases and may particularly be used in the treatment of diseases linked to these kinases, such as e.g. leukemias, lymphomas, solid tumors and autoimmune disorders.

Claims

1. A compound of formula (I): ##STR00355## wherein X.sup.1 is selected from the group consisting of nitro, cyano, methyl, trifluoromethyl, C(O)T.sup.1, C(O)OT.sup.4 and S(O).sub.2T.sup.4; Z and X.sup.2 are each independently selected from the group consisting of F, Cl, Br, and I; X.sup.3 is isopropyl or ethyl; X.sup.4 is either absent or selected from NR.sup.4 and N(R.sup.4)(CH.sub.2); R.sup.4 is selected from H and C.sub.1-6alkyl; Y.sup.1 is selected from the group consisting of H, C.sub.1-6alkyl and a 4- to 7-membered saturated or unsaturated aromatic carbocycle or heterocycle, with the proviso that the point of attachment on said heterocycle is carbon if X.sup.4 is NR.sup.4 or N(R.sup.4)(CH.sub.2), wherein said C.sub.1-6alkyl is optionally substituted with one or more substituents independently selected from OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3) and a 5- to 6-membered saturated heterocycle, wherein said 4- to 7-membered carbocycle or heterocycle is selected from the group consisting of cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, azetidine, oxetane, thietane, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, piperidine, tetrahydropyran, thiane, piperazine, morpholine, thiomorpholine, azepane, oxepane, thiepane, homopiperazine, phenyl, pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, pyrazine, pyrimidine and pyridazine, and wherein said 4- to 7-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3), oxo and C.sub.1-3alkyl, wherein said C.sub.1-3 alkyl is optionally substituted with one or more substituents independently selected from OT.sup.7, N(T.sup.2)(T.sup.3) and a 6-membered saturated heterocycle; T.sup.1, T.sup.2 and T.sup.3 are each independently selected from H and C.sub.1-6alkyl optionally substituted with one or more substituents independently selected from F, N(T.sup.5)(T.sup.6), OT.sup.7, ST.sup.7, cyano, C(O)OT.sup.7, C(O)N(T.sup.5)(T.sup.6), OC(O)N(T.sup.5)(T.sup.6), S(O).sub.2T.sup.7, S(O).sub.2OT.sup.8 and S(O).sub.2N(T.sup.5)(T.sup.6); T.sup.4 is C.sub.1-6alkyl optionally substituted with one or more substituents independently selected from F, N(T.sup.5)(T.sup.6), OT.sup.7, ST.sup.7, cyano, C(O)OT.sup.7, C(O)N(T.sup.5)(T.sup.6), OC(O)N(T.sup.5)(T.sup.6), S(O).sub.2T.sup.8, S(O).sub.2OT.sup.7 and S(O).sub.2N(T.sup.5)(T.sup.6); T.sup.5, T.sup.6 and T.sup.7 are each independently selected from H and C.sub.1-6alkyl optionally substituted with one or more substituents independently selected from F, amino, hydroxyl, thiol and cyano; and T.sup.8 is selected from C.sub.1-6alkyl optionally substituted with one or more substituents independently selected from F, amino, hydroxyl, thiol and cyano; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein X.sup.1 is selected from the group consisting of nitro, cyano, trifluoromethyl, C(O)T.sup.1, and S(O).sub.2T.sup.4; or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, wherein Y.sup.1 is a 4- to 7-membered saturated or unsaturated aromatic carbocycle or heterocycle, with the proviso that the point of attachment on said heterocycle is carbon if X.sup.4 is NR.sup.4 or N(R.sup.4)(CH.sub.2), wherein said 4- to 7-membered carbocycle or heterocycle is selected from the group consisting of cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, azetidine, oxetane, thietane, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, piperidine, tetrahydropyran, thiane, piperazine, morpholine, thiomorpholine, azepane, oxepane, thiepane, homopiperazine, phenyl, pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, pyrazine, pyrimidine and pyridazine, and wherein said 4- to 7-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3), oxo and C.sub.1-3alkyl, wherein said C.sub.1-3alkyl is optionally substituted with one or more substituents independently selected from OT.sup.7, N(T.sup.2)(T.sup.3) and a 6-membered saturated heterocycle; or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 3, wherein Y.sup.1 is a 4- to 7-membered saturated carbocycle or heterocycle, with the proviso that the point of attachment on said heterocycle is carbon if X.sup.4 is NR.sup.4 or N(R.sup.4)(CH.sub.2), wherein said 4- to 7-membered carbocycle or heterocycle is selected from the group consisting of cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, azetidine, oxetane, thietane, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, piperidine, tetrahydropyran, thiane, piperazine, morpholine, thiomorpholine, azepane, oxepane, thiepane, and homopiperazine, and wherein said 4- to 7-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3), oxo and C.sub.1-3alkyl, wherein said C.sub.1-3alkyl is optionally substituted with one or more substituents independently selected from OT.sup.7, N(T.sup.2)(T.sup.3) and a 6-membered saturated heterocycle; or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 3, wherein X.sup.4 is absent; or a pharmaceutically acceptable salt thereof.

6. The compound according to claim 1, wherein X.sup.4 is absent and Y.sup.1 is a 6-membered saturated heterocycle, wherein said 6-membered saturated heterocycle is selected from the group consisting of piperidine, tetrahydropyran, thiane, piperazine, morpholine, and thiomorpholine, and wherein said 6-membered heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3), oxo and C.sub.1-3alkyl; or a pharmaceutically acceptable salt thereof.

7. The compound according to claim 1, wherein said compound is selected from the group consisting of: 5,6-dibromo-1-ethyl-4-nitro-2-(piperazin-1-yl)-1H-1,3-benzodiazole; 5,6-dibromo-4-nitro-2-(piperazin-1-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole; (3 S)-1-(5,6-dibromo-1-ethyl-4-nitro-1H-1,3-benzodiazol-2-yl)piperidin-3-amine; 5,6-dibromo-2-[(2 S)-2-methylpiperazin-1-yl]-4-nitro-1-(propan-2-yl)-1H-1,3-benzodiazole; and 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole.

8. The compound according to claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate.

9. A pharmaceutical composition comprising: a pharmaceutically effective amount of a compound of formula (I): ##STR00356## wherein said compound of formula (I) is a pharmaceutically active agent; and wherein said pharmaceutical composition comprises a pharmaceutically acceptable excipient; wherein X.sup.1 is selected from the group consisting of nitro, cyano, methyl, trifluoromethyl, C(O)T.sup.1, C(O)OT.sup.4 and S(O).sub.2T.sup.4; Z and X.sup.2 are each independently selected from the group consisting of F, Cl, Br, and I; X.sup.3 is selected from the group consisting of C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynyl and a 3- to 6-membered saturated carbocycle or heterocycle, with the proviso that the point of attachment on said heterocycle is carbon, wherein said 3- to 6-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1 and S(O).sub.2N(T.sup.2)(T.sup.3), and wherein said C.sub.1-6alkyl, C.sub.2-6alkenyl and C.sub.2-6alkynyl is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3) and a 3- to 6-membered saturated carbocycle or heterocycle, wherein said 3- to 6-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1 and S(O).sub.2N(T.sup.2)(T.sup.3); X.sup.4 is either absent or selected from NR.sup.4 and N(R.sup.4)(CH.sub.2); R.sup.4 is selected from H and C.sub.1-6alkyl; Y.sup.1 is selected from the group consisting of H, C.sub.1-6alkyl and a 4- to 7-membered saturated or unsaturated aromatic carbocycle or heterocycle, with the proviso that the point of attachment on said heterocycle is carbon if X.sup.4 is NR.sup.4 or N(R.sup.4)(CH.sub.2), wherein said C.sub.1-6alkyl is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3) and a 5- to 6-membered saturated heterocycle, and wherein said 4- to 7-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3), oxo and C.sub.1-3alkyl, wherein said C.sub.1-3alkyl is optionally substituted with one or more substituents independently selected from OT.sup.7, N(T.sup.2)(T.sup.3) and a 6-membered saturated heterocycle; T.sup.1, T.sup.2 and T.sup.3 are each independently selected from H and C.sub.1-6alkyl optionally substituted with one or more substituents independently selected from F, N(T.sup.5)(T.sup.6), OT.sup.7, ST.sup.7, cyano, C(O)OT.sup.7, C(O)N(T.sup.5)(T.sup.6), OC(O)N(T.sup.5)(T.sup.6), S(O).sub.2T.sup.7, S(O).sub.2OT.sup.8 and S(O).sub.2N(T.sup.5)(T.sup.6); T.sup.4 is C.sub.1-6alkyl optionally substituted with one or more substituents independently selected from F, N(T.sup.5)(T.sup.6), OT.sup.7, ST, cyano, C(O)OT.sup.7, C(O)N(T.sup.5)(T.sup.6), OC(O)N(T.sup.5)(T.sup.6), S(O).sub.2T.sup.8, S(O).sub.2OT.sup.7 and S(O).sub.2N(T.sup.5)(T.sup.6); T.sup.5, T.sup.6 and T.sup.7 are each independently selected from H and C.sub.1-6alkyl optionally substituted with one or more substituents independently selected from F, amino, hydroxyl, thiol and cyano; and T.sup.8 is selected from C.sub.1-6alkyl optionally substituted with one or more substituents independently selected from F, amino, hydroxyl, thiol and cyano; or a pharmaceutically acceptable salt thereof, wherein said compound is provided in a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, or wherein said compound is provided as an oral dosage form.

10. A method for treating a disease in a subject, comprising administering to said subject a pharmaceutical composition according to claim 9, wherein said disease is selected from the group consisting of prostate cancer, a leukemia, and a lymphoma.

11. The method of claim 10, wherein said disease is prostate cancer.

12. The method of claim 10, wherein said disease is a leukemia selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, and chronic lymphocytic leukemia.

13. The method of claim 10, wherein said disease is a lymphoma selected from diffuse large B-cell lymphoma.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: PIM-kinase biomarkers in MV4-11 cells upon incubation of the cells with compound 1A of the present invention (see example 3.14 for further details).

(2) FIG. 2: PIM-kinase biomarkers in MV4-11 cells upon incubation of the cells with compound 2A of the present invention (see example 3.14 for further details).

(3) FIG. 3: PIM-kinase biomarkers in MV4-11 cells upon incubation of the cells with compound 1BI of the present invention (see example 3.14 for further details).

(4) FIG. 4: PIM-kinase biomarkers in MOLM-16 cells upon incubation of the cells with compound 1BI of the present invention (see example 3.14 for further details).

(5) FIG. 5: Tumor volume kinetics and body weight kinetics for MOLM16 xenografts with compound 2A (see example 3.15 for further details).

(6) FIG. 6: Tumor volume kinetics and body weight kinetics for MV-4-11 xenografts with compound 26A alone and in combination with Cytarabine (see example 3.15 for further details).

DETAILED DESCRIPTION OF THE INVENTION

(7) The inventors of the present invention inter alia succeeded in identifying new compounds which efficiently inhibit PIM1-3- and DYRK1A-kinases. The compounds of the present invention may thus be particularly used in the treatment of cancer, autoimmune diseases and inflammatory diseases. Before some of the embodiments of the present invention are described in more detail, the following definitions are introduced.

1. DEFINITIONS

General Definitions

(8) As used in the specification and the claims, the singular forms of a and an also include the corresponding plurals unless the context clearly dictates otherwise. The same applies for plural forms used herein, which also include the singular forms unless the context clearly dictates otherwise.

(9) The terms about and approximately in the context of the present invention denotes an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of 10% and preferably 5%.

(10) It needs to be understood that the term comprising is not limiting. For the purposes of the present invention, the term consisting of is considered to be a preferred embodiment of the term comprising of. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also meant to encompass a group which preferably consists of these embodiments only.

(11) The term alkyl refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C.sub.1-6 indicates that the group can have from 1 to 6 (inclusive) carbon atoms in it. If there is no indication of carbon atoms of the alkyl, the term alkyl refers to a C.sub.1-15alkyl, preferably a C.sub.1-10alkyl, and more preferably to a C.sub.1-4alkyl.

(12) In general, the number of carbon atoms present in a given group is designated C.sub.x-y where x and y are the lower and upper limits, respectively. For example, a group designated as C.sub.1-5 contains from 1 to 5 (inclusive) carbon atoms. The carbon number as used in the definitions herein refers to carbon backbone and carbon branching, but does not include carbon atoms of the substituents. General examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, n-butyl, isobutyl, secbutyl, tert-butyl, and pentyl. For example, the term C.sub.1-3alkyl refers to a straight or branched chain saturated hydrocarbon containing 1-3 carbon atoms. Examples of a C.sub.1-3alkyl group include, but are not limited to, methyl, ethyl, propyl and isopropyl. For example, the term C.sub.6-10alkyl refers to a straight or branched chain saturated hydrocarbon containing 6-10 carbon atoms. Examples of a C.sub.6-10alkyl group include, but are not limited to, hexyl, octyl and decyl.

(13) Alkenyl is a hydrocarbon chain having at least one (preferably only one) carbon-carbon double bond. Alkynyl is a hydrocarbon chain having at least one (preferably only one) carbon-carbon triple bond.

(14) The term heterocycle refers to a cyclic structure comprising carbon atoms and at least one heteroatom. The term heteroatom as used herein preferably refers to nitrogen, sulfur and oxygen atoms. A heterocycle may generally contain different heteroatoms. For the present invention, nitrogen as heteroatom may be preferred. Further, for the present invention, it can be preferred that a heterocycle comprises one or two heteroatoms. If reference to a specific heterocycle is made herein (such as e.g. to piperazine), this reference has to be understood as relating to the commonly used and defined structure of said heterocycle in the field of chemistry.

(15) If e.g. reference to a 4- to 7-membered saturated or unsaturated aromatic carbocycle or heterocycle is made herein, it needs to be understood that the term aromatic is used in combination with the term unsaturated only; thus, the above definition may also be regarded as short definition of a 4- to 7-membered saturated non-aromatic or a 4- to 7-membered unsaturated aromatic carbocycle or heterocycle. Of course, the term aromatic as used in the short definition is not to be read in combination with the term saturated since reference would otherwise be made to a non-existing saturated aromatic carbocycle or heterocycle.

(16) The term halogen includes fluorine, bromine, chlorine or iodine. The term amino represents NH.sub.2, the term hydroxyl is OH, the term thiol is SH, the term nitro is NO.sub.2, the term cyano is CN and oxo is O. Carbon branching or branched alkyl means that one or more alkyl groups such as methyl, ethyl or propyl, replace one or both hydrogens in a CH.sub.2 group of a linear alkyl chain.

(17) If a substituent is not defined as the final substituent but rather as a bridging substituent (such as e.g. the X.sup.4 definition of NR.sup.4(CH.sub.2)), the definition is preferably used in terms of the orientation in a compound of the present invention as from left to right in the overall structure. This means e.g. for NR.sup.4(CH.sub.2) that the nitrogen is attached to the benzimidazole-moiety, whereas the CH.sub.2 is attached to substituent Y.sup.1.

(18) If a point of attachment on a heterocycle is referred to herein, this refers to an atom in the heterocycle, to which the remaining moiety of the compound is attached to. In some cases of the present invention, this may refer to the attachment of X.sup.4 to a heterocycle in the Y.sup.1-position or, alternatively, if X.sup.4 is not present, to the attachment of the benzimidazole-moiety at position 2 to the heterocycle in the Y.sup.1-position (direct bond). In other cases of the present invention, this may refer to the attachment of a heterocycle in the X.sup.3-position to the nitrogen-atom of the benzimidazole-moiety.

(19) The invention disclosed herein is meant to encompass all pharmaceutically acceptable salts of the disclosed compounds, particularly the salts referred to above. Further, the pharmaceutically acceptable salts include metal salts such as sodium salt, potassium salt, cesium salt and the like; alkaline earth metals such as calcium salt, magnesium salt and the like; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N-dibenzylethylenediamine salt and the like; inorganic acid salts such as hydrochloride, hydrobromide, sulfate, phosphate and the like; organic acid salts such as formate, acetate, trifluoroacetate, maleate, fumarate, tartrate and the like; sulfonates such as methanesulfonate, benzenesulfonate, p-toluenesulfonate, and the like; amino acid salts such as arginate, asparginate, glutamate and the like. A particularly preferred pharmaceutically acceptable salt may be selected from the group consisting of the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate. The hydrochloride salt is particularly preferred for compounds of the present invention.

(20) The compounds disclosed herein may contain one or more asymmetric centers and may thus lead to enantiomers, diastereomers, and other stereoisomeric forms. The present invention is also meant to encompass all such possible forms as well as their racemic and resolved forms and mixtures thereof, unless specified otherwise. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended to include both E and Z geometric isomers. All tautomers are intended to be encompassed by the present invention as well.

(21) As used herein, the term stereoisomers is a general term for all isomers of individual molecules that differ only in the orientation of their atoms in space. It includes enantiomers and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereomers). The term chiral center refers to an atom to which four different groups are attached. The term enantiomer or enantiomeric refers to a molecule that is nonsuperimposeable on its mirror image and hence optically active wherein the enantiomer rotates the plane of polarized light in one direction and its mirror image rotates the plane of polarized light in the opposite direction. The term racemic refers to a mixture of equal parts of enantiomers and which is optically inactive. The term resolution refers to the separation or concentration or depletion of one of the two enantiomeric forms of a molecule.

(22) Pharmaceutically active agent as used herein means that a compound is potent of modulating a response in a human or animal being in vivo. When reference is made to a compound as the only pharmaceutically active agent, this is meant to describe that the activity of a corresponding pharmaceutical composition is due to said active agent only.

(23) The term pharmaceutically acceptable excipient as used herein refers to compounds commonly comprised in pharmaceutical compositions, which are known to the skilled person. Such compounds or excipients are exemplary listed below. In view of the definition pharmaceutically active agent as given above, a pharmaceutically acceptable excipient can be defined as being pharmaceutically inactive.

(24) Description of Pharmaceutical Compositions According to the Present Invention

(25) A pharmaceutical composition according to the present invention may be formulated for oral, buccal, nasal, rectal, topical, transdermal or parenteral application. Oral application may be preferred. Parenteral application can also be preferred and includes intravenous, intramuscular or subcutaneous administration. The compound according to formula (I) should be applied in pharmaceutically effective amounts, for example in the amounts as set out herein below.

(26) A pharmaceutical composition of the present invention may also be designated as formulation or dosage form. A compound of formula (I) may also be designated in the following as (pharmaceutically) active agent or active compound.

(27) Pharmaceutical compositions may be solid or liquid dosage forms or may have an intermediate, e.g. gel-like character depending inter alia on the route of administration.

(28) In general, the inventive dosage forms can comprise various pharmaceutically acceptable excipients which will be selected depending on which functionality is to be achieved for the dosage form. A pharmaceutically acceptable excipient in the meaning of the present invention can be any substance used for the preparation of pharmaceutical dosage forms, including coating materials, film-forming materials, fillers, disintegrating agents, release-modifying materials, carrier materials, diluents, binding agents and other adjuvants. Typical pharmaceutically acceptable excipients include substances like sucrose, mannitol, sorbitol, starch and starch derivatives, lactose, and lubricating agents such as magnesium stearate, disintegrants and buffering agents.

(29) The term carrier denotes pharmaceutically acceptable organic or inorganic carrier substances with which the active ingredient is combined to facilitate the application. Suitable pharmaceutically acceptable carriers include, for instance, water, salt solutions, alcohols, oils, preferably vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, surfactants, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and the like. The pharmaceutical compositions can be sterilized and if desired, mixed with auxiliary agents, like lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compound.

(30) If liquid dosage forms are considered for the present invention, these can include pharmaceutically acceptable emulsions, solutions, suspensions and syrups containing inert diluents commonly used in the art such as water. These dosage forms may contain e.g. microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer and sweeteners/flavouring agents.

(31) For parenteral application, particularly suitable vehicles consist of solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. Pharmaceutical formulations for parenteral administration are particularly preferred and include aqueous solutions of the compounds of formula (I) in water-soluble form. Additionally, suspensions of the compounds of formula (I) may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

(32) Particularly preferred dosage forms are injectable preparations of a compound of formula (I). Thus, sterile injectable aqueous or oleaginous suspensions can for example be formulated according to the known art using suitable dispersing agents, wetting agents and/or suspending agents. A sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluant or solvent. Among the acceptable vehicles and solvents that can be used are water and isotonic sodium chloride solution. Sterile oils are also conventionally used as solvent or suspending medium.

(33) Suppositories for rectal administration of a compound of formula (I) can be prepared by e.g. mixing the compound with a suitable non-irritating excipient such as cocoa butter, synthetic triglycerides and polyethylene glycols which are solid at room temperature but liquid at rectal temperature such that they will melt in the rectum and release the compound according to formula (I) from said suppositories.

(34) For administration by inhalation, the compounds according to the present invention may be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

(35) Oral dosage forms may be liquid or solid and include e.g. tablets, troches, pills, capsules, powders, effervescent formulations, dragees and granules. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. The oral dosage forms may be formulated to ensure an immediate release of the compound of formula (I) or a sustained release of the compound of formula (I).

(36) A solid dosage form may comprise a film coating. For example, the inventive dosage form may be in the form of a so-called film tablet. A capsule of the invention may be a two-piece hard gelatin capsule, a two-piece hydroxypropylmethylcellulose capsule, a two-piece capsule made of vegetable or plant-based cellulose or a two-piece capsule made of polysaccharide.

(37) The dosage form according to the invention may be formulated for topical application. Suitable pharmaceutical application forms for such an application may be a topical nasal spray, sublingual administration forms and controlled and/or sustained release skin patches. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

(38) The compositions may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. The methods can include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Liquid dose units are vials or ampoules. Solid dose units are tablets, capsules and suppositories.

(39) As regards human patients, the compound of formula (I) may be administered to a patient in an amount of about 0.001 mg to about 5000 mg per day, preferably of about 0.01 mg to about 100 mg per day, more preferably of about 0.1 mg to about 50 mg per day.

(40) Indications, for which the Compounds of the Present Invention May be Used

(41) The compounds according to the present invention may be used for the treatment of a disease selected from the group consisting of myeloid leukemia (both acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome, Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma); adenocarcinoma, lymphoma, leukemia of the kidney, Wilm's tumor, renal cell carcinoma, renal pelvis carcinoma, nephroma, teratoma, sarcoma of the kidney, squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma of bladder and urethra, sarcoma of the prostate, seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma of the testis; angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma of the heart; astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors of the brain, neurofibroma, meningioma, glioma, sarcoma of the spinal cord, osteoma, hemangioma, granuloma, xanthoma, osteitis deformians of the skull, meningioma, meningiosarcoma, gliomatosis of the meninges; undifferentiated small cell squamous cell, undifferentiated large cell squamous cell, adenocarcinoma, alveolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hanlartoma, mesothelioma of the bronchus; lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma of the small bowel, adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma of the large bowel; squamous cell carcinoma, leiomyosarcoma, lymphoma of the esophagus, lymphoma, leiomyosarcoma of the stomach, ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma of the pancreas; hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma of the liver; osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma such as reticulum cell sarcoma, multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma such as osteocartilaginous exostoses, benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; endometrial carcinoma, cervical carcinoma, pre-tumor cervical dysplasia, ovarian carcinoma such as serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa-thecal cell tumors, Sertol/Leydig cell tumors, dysgerminoma, malignant teratoma of the ovary, squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma of the vulva, clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma such as embryonal rhabdomyosarcoma of the vagina, fallopian tubes carcinoma), breast; and malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, bone marrow transplant rejection, rheumatoid arthritis, psoriasis, type I diabetes mellitus and multiple sclerosis.

(42) Since the compounds of the present invention are PIM-kinase inhibitors, they may particularly be used for the treatment of PIM-kinase linked diseases. Thus, the compounds of the present invention may be used for the treatment of cancer, in particular hematopoietic malignancies such as diffuse B cell lymphoma, chronic lymphocytic leukemia and acute myelogenous leukemia, follicular lymphoma (FL) and B-chronic lymphocytic leukemia (B-CLL), diffuse large B-cell lymphoma (DLBCL), AIDS-associated non-Hodgkin lymphoma, HCV-infected B-cell NHL, primary central nervous system lymphomas (PCNSLs), extranodal DLBCL, primary cutaneous marginal zone B-cell lymphoma (PCMZL), primary mediastinal large B-cell lymphoma (PMLBCL); acute myeloid leukemias (AML); chronic myelogenous leukemia; invasive head and neck squamous cell carcinomas (HNSCC); solid tumors such as prostate cancer, pancreatic cancer, gastrointestinal cancer, colon cancer, liver cancer; and hepatocellular carcinoma (HCC). Further, the compounds may be used for the treatment of an inflammatory disease, in particular rheumatoid arthritis, lupus, multiple sclerosis and inflammatory bowel disease.

(43) Some compounds of the present invention not only inhibit PIM-kinases but also the FLT3-kinase. When reference is made in the present application to the FLT3-kinase, this is meant to include mutant versions thereof. FLT3 (FMS-like tyrosine kinase) plays a crucial role in pathogenesis of acute myeloid leukemia (AML) which is most common type of acute leukemias in adults and in 20% of childhood leukemia cases. Inhibition of FLT3 kinase, which is frequently overexpressed and mutated (e.g. ITD mutation which is usually associated with poor prognosis) in AML patients is a promising target for the therapy. In addition to inhibition FLT3 itself which should be beneficial in AML treatment, combination of inhibitory activity against FLT3 and PIM which are on the same signaling pathway should be an especially desired way of acting against hematological malignancies helping e.g. overcoming drug resistance. Thus, compounds according to the present invention inhibiting PIM-kinases and the FLT3-kinase may particularly be used in the treatment of AML; it can be especially preferred to treat AML patients harbouring an ITD mutation, D835H, D835Y or N8411 in FLT3 with such compounds.

(44) A role of DYRK1 in cancer is described. DYRK1A potentiates the transcriptional activity Gli1 (glioma-associated oncogene homologue 1), a transcription factor being a terminal effector of hedgehog signaling, which is a key pathway for embryogenesis, stem cell maintenance and tumorigenesis (J. Med. Chem., 2009, 52(13), 3829-3845). DYRK1A acts as a negative regulator of apoptosis. (FEBS J., 2008, 275(24), 6268-6280) Therefore inhibiting DYRK1A activity in cancer cells was proposed as a new strategy to combat the dismal prognosis associated with cancers that display resistance to pro-apoptotic stimuli. STAT3, that is overexpressed in various cancers and represents an interesting target to impede cancer progression, is also activated by DYRK1A (Curr Cancer Drug Targets. 2010 February; 10(1):117-26; Anticancer Agents Med Chem. 2010 September; 10(7):512-9.). The compounds of the present invention may thus be used in order to treat cancer, in particular glioblastoma, breast cancer, gliomas, melanomas, esophageal cancer, pancreas cancer and non-small-cell lung cancers.

(45) DYRK1A is also believed to be implicated in neural differentiation. (Neurobiol Dis. 2012 April; 46(1):190-203) The role of DYRK1A kinase in neurodegeneration is well established, therefore Alzheimer's disease, Down syndrome and other taupathies such as progressive supranuclear palsy, Pick's disease, chronic traumatic encephalopathy and frontotemporal dementia may also be treated with the compounds according to the present application. (FEBS J. 2011 January; 278(2):236-45; J Neuropathol Exp Neurol. 2011 January; 70(1):36-50). DYRK1A kinase was also associated with development of pathology in -synuclein dementias such as dementia with Lewy bodies and Parkinson's disease dementia (J Biol Chem. 2006 Nov. 3; 281(44):33250-7; Neurodegener Dis. 2012; 10(1-4):229-31).

(46) Most preferably, the compounds of the present invention may be used for the treatment of a disease selected from the group consisting of leukemias including acute lymphoblastic leukemia, acute myelogenous leukemia and chronic lymphocytic leukemia, lymphoma, myeloma, myloproliferative disorder, allograft rejection, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Alzheimer disease and Down syndrome.

(47) In a preferred embodiment relating to the pharmaceutical compositions of the present invention, said pharmaceutical composition comprises said compound as the only pharmaceutically active agent.

(48) Alternatively, said pharmaceutical composition comprises at least one further independent pharmaceutically active agent in addition to said compound. As outlined above, the pharmaceutical composition according to the present invention may particularly be used in the treatment of cancer, an autoimmune or an inflammatory disease or neurodegenerative disorders such that at least one further independent pharmaceutically active agents directed to the treatment of such a particular disease may be additionally present.

(49) Further, the compounds of the present invention may be useful as adjuvants to e.g. cancer treatment. They may be used in combination with one or more additional drugs, for example a chemotherapeutic agent which acts by the same or by a different mechanism of action. Such drugs are listed in the example section of the present application and comprise both targeted agents such as kinase inhibitors of the PI3K/Akt/mTOR pathway or the JAK/STAT pathway, but also standard chemotherapy agents such as cytarabine, and vosaroxin. In particular, the compounds of preferred embodiments (A) stated above may be used in cancer therapy (e.g. for use in treating acute myelogenous leukemia (AML), diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM)) in combination with a chemotherapeutic agent such as a PI3K inhibitor, a JAK kinase inhibitor, cytarabine, vosaroxin and combinations thereof. Other targeted cancer therapy agents such as e.g. kinase inhibitors may, however, also be used in combination with compounds of the present invention.

2. ALTERNATIVE FORMULATIONS

(50) The subject matter of the present invention may also be referred to as follows:

(51) Method of administering to a subject in need thereof an effective amount of a compound according to formula (I) or a pharmaceutically acceptable salt thereof as defined above (including the preferred embodiments).

(52) Method of treating a disease selected from the disease as disclosed herein by administering to a subject in need thereof an effective amount of a compound according to formula (I) or a pharmaceutically acceptable salt thereof as defined above (including the preferred embodiments).

(53) Method for treating a PIM1-3- and/or FLT3- and/or DYRK1A-related disorder, said method comprising the step of administering to a patient in need thereof a therapeutic amount of a compound according to formula (I) or a pharmaceutically acceptable salt thereof as defined above (including the preferred embodiments).

(54) Method for treating a PIM1-3- and/or FLT3- and/or DYRK1A-related cancer, said method comprising the step of administering to a patient in need thereof a therapeutic amount of a compound according to formula (I) or a pharmaceutically acceptable salt thereof as defined above (including the preferred embodiments).

(55) Method for treating a PIM1-3- and/or FLT3- and/or DYRK1A-related inflammatory disorder, said method comprising the step of administering to a patient in need thereof a therapeutic amount of a compound according to formula (I) or a pharmaceutically acceptable salt thereof as defined above (including the preferred embodiments).

(56) Method for treating a PIM1-3- and/or FLT3- and/or DYRK1A-related autoimmune disorder, said method comprising the step of administering to a patient in need thereof a therapeutic amount of a compound according to formula (I) or a pharmaceutically acceptable salt thereof as defined above (including the preferred embodiments).

(57) Method for treating a PIM1-3- and/or FLT3- and/or DYRK1A-related neurodegenerative disorder, said method comprising the step of administering to a patient in need thereof a therapeutic amount of a compound according to formula (I) or a pharmaceutically acceptable salt thereof as defined above (including the preferred embodiments).

(58) In the following, examples of embodiments of the present invention are outlined. However, said examples should not be construed as limiting the scope of the present invention.

3. EXAMPLES

3.1. Compounds of Example 1

(59) ##STR00003##

5,6-dibromo-1-ethyl-4-nitro-2-(piperazin-1-yl)-1H-1,3-benzodiazole hydrochloride (Example 1A)

(60) 2,5,6-tribromo-1-ethyl-4-nitro-1H-1,3-benzodiazole (150 mg, 0.35 mmol) and BOC piperazine (260 mg, 1.4 mmol) was dissolved in EtOH (3.0 ml). The resulting mixture was stirred at temperature 170 C. under microwave conditions until the reaction was completed (20 min) by LC/MS. The mixture was allowed to cool to RT and concentrated in-vacuo. The product was purified on silica gel using EA/hex (1:1). The product was dissolved in 1,4-dioxane (3.0 ml) and 4M HCl in dioxane (1.0 ml) was added. The mixture was stirred at room temperature until the reaction was complete (18 hrs) by LC/MS. Diethyl ether (5.0 ml) was added, product was filtered off, washed with diethyl ether and dried to afford 5,6-dibromo-1-ethyl-4-nitro-2-(piperazin-1-yl)-1H-1,3-benzodiazole hydrochloride (41 mg, 0.087 mmol). 1H NMR (600 MHz, DMSO) 9.59 (s, 1H), 8.21 (s, 1H), 4.18 (q, J=7.2 Hz, 2H), 3.60-3.58 (m, 4H), 3.25 (s, 4H), 1.33 (t, J=7.2 Hz, 3H); m/z 433.8; rt 2.4 min.

(61) The following compounds were prepared by the procedure of Example 1A, using the appropriate starting materials (SM):

(62) TABLE-US-00001 1HNMR Ex. Name and structure (400 MHz) m/z rt SM 1C 1H NMR (600 MHz, DMSO) 8.17 (s, 4H), 8.04 (s, 2H), 7.93 (s, 1H), 4.16 (q, J = 7.1 Hz, 2H), 3.46 (dd, J = 11.6, 6.0 Hz, 2H), 2.88 (tt, J = 13.3, 6.5 Hz, 2H), 1.95- 1.87 (m, 2H), 1.19 (t, J = 7.1 Hz, 3H). 421.8 2.5 1D 1H NMR (600 MHz, DMSO) 8.43 (s, 1H), 7.56 (s, 1H), 3.38 (t, J = 6.5 Hz, 2H), 2.84 (t, J = 7.1 Hz, 2H), 1.81 (p, J = 6.8 Hz, 2H). 393.8 2.2 1E 1H NMR (600 MHz, DMSO) 9.41 (bs, 1H), 7.72 (s, 1H), 3.84 (dd, J = 15.7, 10.4 Hz, 4H), 3.22 (bs, 4H). 405.8 2.4 1F 0 .sup.1H NMR (600 MHz, DMSO) 8.42 (bs, 3H), 8.17 (s, 1H), 4.17 (tt, J = 13.8, 7.0 Hz, 2H), 3.79 (dd, J = 12.4, 3.4 Hz, 1H), 3.51- 3.46 (m, 1H), 3.36 (d, J = 4.4 Hz, 1H), 3.18 (dd, J = 12.5, 9.0 Hz, 1H), 3.10-3.04 (m, 1H), 2.04 (dd, J = 8.8, 3.9 Hz, 1H), 1.91 (dd, J = 8.8, 4.3 Hz, 1H), 1.73- 1.61 (m, 2H), 1.35 (t, J = 7.2 Hz, 3H). 447.9 2.4 1G 1H NMR (600 MHz, DMSO) 7.89 (s, 1H), 4.15-4.07 (m, 3H), 3.84- 3.78 (m, 1H), 3.44-3.36 (m, 4H), 2.78 (dd, J = 12.9, 3.8 Hz, 1H), 2.63- 2.58 (m, 1H), 1.19 (t, J = 7.1 Hz, 3H), 1.07 (s, 1H). 437.9 2.4 1H 461.9 2.7 1I 447.9 2.6 1L .sup.1H NMR (600 MHz, DMSO) 8.05 (bs, 3H), 7.94 (s, 1H), 7.86 (bs, 1H), 4.79 (hept, J = 6.9 Hz, 1H), 3.46 (bs, 2H), 2.89- 2.83 (m, 2H), 1.94-1.88 (m, 2H), 1.49 (d, J = 6.9 Hz, 6H). 435.9 2.6 1M 0 .sup.1H NMR (600 MHz, DMSO) 9.62 (s, 2H), 8.26 (s, 1H), 4.13-4.08 (m, 2H), 3.60- 3.57 (m, 4H), 3.24 (s, 4H), 1.78-1.70 (m, 2H), 0.85 (t, J = 7.4 Hz, 3H). 447.9 2.5 1N .sup.1H NMR (600 MHz, DMSO) 9.55 (bs, 2H), 8.32 (s, 1H), 4.02 (d, J = 7.6 Hz, 2H), 3.59-3.54 (m, 4H), 3.23 (bs, 4H), 2.22- 2.12 (m, 1H), 0.78 (d, J = 6.6 Hz, 6H). 461.9 2.8 1P .sup.1H NMR (600 MHz, DMSO) 8.10 (bs, 1H), 8.05 (bs, 2H), 7.95 (s, 1H), 6.74 (bs, 1H), 4.08 (t, J = 7.4 Hz, 2H), 3.47 (dd, J = 11.8, 6.1 Hz, 2H), 2.89- 2.83 (m, 2H), 1.93-1.87 (m, 2H), 1.67- 1.60 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). 418.9 3.6 1Q 440.9 3.8 1R 1H NMR (600 MHz, DMSO) 9.42 (s, 1H), 8.30 (s, 1H), 4.09 (d, J = 7.0 Hz, 2H), 3.56 (dd, J = 16.8, 11.6 Hz, 4H), 3.28-3.21 (m, 4H), 1.30- 1.24 (m, 1H), 0.51-0.44 (m, 2H), 0.41- 0.37 (m, 2H). 445.9 2.7 1S 0 1H NMR (600 MHz, DMSO) 8.28 (s, 3H), 7.67 (s, 1H), 4.19 (dd, J = 12.7, 3.3 Hz, 1H), 3.79 (dt, J = 12.7, 4.2 Hz, 1H), 3.39- 3.25 (m, 3H), 2.05-1.99 (m, 1H), 1.85 (dd, J = 9.3, 3.9 Hz, 1H), 1.70- 1.57 (m, 2H). 461.9 2.8 1T .sup.1H NMR (600 MHz, DMSO) 8.40 (bs, 3H), 8.25 (s, 1H), 4.60 (hept, J = 6.8 Hz, 1H), 3.59 (dd, J = 12.3, 3.2 Hz, 1H), 3.39 (d, J = 2.7 Hz, 1H), 3.27 (dd, J = 8.5, 4.4 Hz, 1H), 3.15 (dd, J = 12.3, 8.7 Hz, 1H), 3.01 (dd, J = 15.8, 6.3 Hz, 1H), 2.02 (dd, J = 9.1, 3.6 Hz, 1H), 1.92 (dd, J = 9.6, 3.9 Hz, 1H), 1.74- 1.61 (m, 2H), 1.55 (dd, J = 10.8, 6.9 Hz, 6H). 461.9 2.5 IU 475.9 2.9 IV 1H NMR (600 MHz, DMSO) 8.37 (d, J = 3.6 Hz, 3H), 8.29 (s, 1H), 4.05-3.96 (m, J = 22.2, 14.6, 7.2 Hz, 2H), 3.89-3.80 (m, 1H), 3.54 (d, J = 12.9 Hz, 1H), 3.36- 3.23 (m, 1H), 3.07 (dd, J = 12.3, 9.8 Hz, 1H), 3.00- 2.93 (m, 1H), 2.18 (dp, J = 13.9, 6.8 Hz, 1H), 2.11- 2.03 (m, 1H), 1.93-1.83 (m, 1H), 1.70- 1.54 (m, 2H), 0.77 (dd, J = 6.3 Hz, 5H). 427.8 2.8 IW 451.8 5.2 IX 0 465.9 6.3 IY 1H NMR (600 MHz, DMSO) 7.97 (s, 1H), 4.03-3.99 (m, 1H), 3.99- 3.95 (m, 2H), 3.49 (dt, J = 13.5, 5.9 Hz, 1H), 3.42- 3.36 (m, 1H), 2.95 (ddd, J = 12.8, 5.9, 3.3 Hz, 1H), 2.78- 2.70 (m, 1H), 2.11 (dp, J = 14.1, 6.9 Hz, 1H), 0.88 (dd, J = 6.6, 2.8 Hz, 6H). 447.9 2.7 1Z 1H NMR (600 MHz, DMSO) 8.14 (s, 3H), 8.05 (s, 1H), 4.39 (dd, J = 11.4, 6.0 Hz, 1H), 4.31 (dq, J = 14.4, 7.1 Hz, 1H), 4.15 (dq, J = 14.3, 7.0 Hz, 1H), 3.88 (dd, J = 15.4, 7.2 Hz, 1H), 3.65 (dd, J = 9.8, 6.3 Hz, 1H), 3.10- 3.01 (m, 2H), 2.10-1.99 (m, 2H), 1.96- 1.88 (m, 2H), 1.34 (t, J = 7.1 Hz, 3H). 457.9 2.2 1AA 481.9 2.2 1AB 1H NMR (600 MHz, DMSO) 8.03 (bs, 3H), 7.97 (s, 1H), 4.07 (d, J = 7.1 Hz, 2H), 3.49-3.44 (m, 2H), 2.90- 2.83 (m, 2H), 1.91 (p, 2H), 1.26-1.18 (m, 1H), 0.48- 0.42 (m, 4H). 430 2.1 1AC 0 447.9 2.6 1AD 1H NMR (600 MHz, DMSO) 9.57 (bs, 1H), 9.36 (bs, 1H), 8.30 (s, 1H), 4.27- 4.13 (m, 2H), 3.87 (td, J = 6.6, 3.6 Hz, 1H), 3.47 (dt, J = 13.6, 5.1 Hz, 1H), 3.38 (dt, J = 13.6, 5.2 Hz, 1H), 3.36- 3.31 (m, 1H), 3.24-3.19 (m, 2H), 3.06 (dt, J = 10.5, 6.5 Hz, 1H), 1.31 (t, J = 7.2 Hz, 3H), 1.16 (d, J = 6.6 Hz, 3H). 448.8 3.2 1AE 433.8 2.7 1AF 1H NMR (600 MHz, DMSO) 7.96 (s, 1H), 4.57 (s, 1H), 4.20 (q, J = 7.2 Hz, 2H), 3.46- 3.37 (m, 2H), 3.31-3.21 (m, 2H), 2.25 (dt, J = 15.1, 7.3 Hz, 1H), 2.08 (td, J = 13.2, 5.8 Hz, 1H), 1.20 (t, J = 7.1 Hz, 3H). 434.8 3 1AG 1H NMR (600 MHz, DMSO) 8.56 (s, 1H), 8.03 (s, 1H), 4.35-4.20 (m, 1H), 3.95 (dd, J = 9.6, 7.8 Hz, 1H), 3.93- 3.88 (m, 1H), 3.85-3.75 (m, 1H), 2.32 (td, J = 13.7, 8.2 Hz, 1H), 2.17 (ddd, J = 16.3, 7.6, 4.3 Hz, 1H), 1.33 (t, J = 7.2 Hz, 1H). 447.8 2.7 1AH 0 447.9 2.6 1AI .sup.1H NMR (600 MHz, DMSO) 8.36 (bs, 3H), 8.17 (s, 1H), 4.21- 4.13 (m, 2H), 3.78 (dd, J = 12.4, 3.3 Hz, 1H), 3.51- 3.45 (m, 1H), 3.40-3.33 (m, 1H), 3.20- 3.15 (m, 1H), 3.10-3.05 (m, 1H), 2.04 (dd, J = 8.9, 3.4 Hz, 1H), 1.91 (dd, J = 9.3, 4.0 Hz, 1H), 1.73- 1.61 (m, 2H), 1.35 (t, J = 7.2 Hz, 3H). 433.8 2.5 1AJ 1H NMR (600 MHz, DMSO) 8.45 (bs, 3H), 8.02 (s, 1H), 4.33- 4.22 (m, 2H), 3.95-3.87 (m, 3H), 3.79 (dt, J = 9.1, 4.9 Hz, 2H), 2.32 (td, J = 13.9, 8.3 Hz, 1H), 2.15 (ddd, J = 12.2, 7.6, 4.1 Hz, 1H), 1.32 (t, J = 7.2 Hz, 3H). 434.8 2.9 1AK 407.9 2.5 1AL 1H NMR (600 MHz, DMSO) 8.15 (s, 3H), 8.06 (t, J = 5.2 Hz, 1H), 7.95 (s, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.63 (q, J = 5.8 Hz, 2H), 3.11-3.04 (m, 2H), 1.22 (t, J = 7.1 Hz, 3H). 447.9 2.8 1AM 0 .sup.1H NMR (600 MHz, DMSO) 9.43 (bs, 1H), 9.15 (bs, 1H), 8.17 (t, J = 5.6 Hz, 1H), 7.97 (s, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.80 (td, J = 13.5, 6.6 Hz, 1H), 3.25-3.19 (m, 1H), 3.19- 3.13 (m, 1H), 2.04 (td, J = 12.6, 7.6 Hz, 1H), 1.97- 1.90 (m, 1H), 1.90-1.82 (m, 1H), 1.72 (dq, J = 12.9, 8.1 Hz, 1H), 1.23 (t, J = 7.1 Hz, 3H). 462.8 3.3 1AN 1H NMR (600 MHz, DMSO) 8.02 (s, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.70- 3.66 (m, 4H), 3.08-3.05 (m, 2H), 2.93- 2.90 (m, 2H), 1.91 (dt, J = 11.5, 5.9 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H). 461.9 3.1 1AO 1H NMR (600 MHz, DMSO) 8.95 (bs, 2H), 8.39 (s, 1H), 4.80 (hept, J = 6.8 Hz, 1H), 3.69- 3.63 (m, 1H), 3.53 (d, J = 14.4 Hz, 1H), 3.30 (d, J = 13.1 Hz, 2H), 3.27-3.17 (m, 2H), 3.05 (dd, J = 12.6, 8.5 Hz, 1H), 1.58 (d, J = 7.0 Hz, 3H), 1.51 (d, J = 6.9 Hz, 3H), 1.05 (d, J = 6.5 Hz, 3H). 461.9 2.8 1AP 1H NMR (300 MHz, dmso) 8.35 (s, 3H), 8.24 (s, 1H), 4.57 (dt, J = 13.8, 6.9 Hz, 1H), 3.61- 3.51 (m, 1H), 3.37 (s, 1H), 3.25 (d, J = 13.0 Hz, 1H), 3.12 (dd, J = 12.2, 8.6 Hz, 1H), 3.04- 2.92 (m, 1H), 1.99 (d, J = 11.6 Hz, 1H), 1.89 (s, 1H), 1.65 (d, J = 8.5 Hz, 1H), 1.53 (dd, J = 6.8, 461.9 2.8 5.4 Hz, 6H). 1AQ 1H NMR (600 MHz, DMSO) 8.28 (s, 1H), 8.15 (s, 3H), 4.66-4.60 (m, 1H), 4.00- 3.97 (m, 1H), 3.96-3.91 (m, 1H), 3.79 (td, J = 11.5, 2.3 Hz, 1H), 3.44 (d, J = 12.4 Hz, 1H), 3.34 (d, J = 11.3 Hz, 1H), 3.09 (ddd, J = 11.3, 8.1, 3.6 Hz, 2H), 2.94 (dd, J = 12.5, 10.4 Hz, 1H), 2.92- 2.86 (m, 1H), 1.58 (d, J = 6.9 Hz, 3H), 1.53 (d, J = 6.9 Hz, 3H). 477.8 5.7 1AR 0 461.9 3 1AS .sup.1H NMR (600 MHz, DMSO) 8.76 (bs, 1H), 8.70 (bs, 1H), 7.98 (s, 1H), 7.33 (d, J = 7.2 Hz, 1H), 4.69 (hept, J = 6.9 Hz, 1H), 4.13 (qd, J = 10.5, 5.4 Hz, 1H), 3.43 (d, J = 11.2 Hz, 1H), 3.20 (d, J = 12.5 Hz, 1H), 2.89- 2.79 (m, 2H), 2.04-1.97 (m, 1H), 1.91 (dd, J = 14.2, 3.6 Hz, 1H), 1.74- 1.61 (m, 2H), 1.50 (dd, J = 6.9, 1.0 Hz, 6H). 461.8 2.9 1AT 1H NMR (600 MHz, DMSO) 8.38 (d, J = 3.5 Hz, 3H), 8.26 (s, 1H), 4.64-4.56 (m, 1H), 3.59 (dd, J = 12.3, 3.2 Hz, 1H), 3.39 (d, J = 4.5 Hz, 1H), 3.27 (dd, J = 8.3, 4.5 Hz, 1H), 3.14 (dd, J = 12.4, 8.7 Hz, 1H), 3.01 (dd, J = 15.8, 6.3 Hz, 1H), 2.02 (dd, J = 8.9, 3.5 Hz, 1H), 1.92 (dd, J = 9.5, 3.9 Hz, 1H), 1.75- 1.61 (m, 2H), 1.55 (dd, J = 10.7, 6.9 Hz, 6H). 475.9 3 1AU 1H NMR (600 MHz, DMSO) 8.31 (d, J = 3.6 Hz, 3H), 8.29 (s, 1H), 4.02 (d, J = 2.7 Hz, 2H), 3.84 (d, J = 3.5 Hz, 1H), 3.82 (d, J = 3.5 Hz, 1H), 3.54 (d, J = 12.9 Hz, 1H), 3.31 (dd, J = 9.4, 4.4 Hz, 1H), 3.06 (dd, J = 12.3, 9.8 Hz, 1H), 3.00- 2.92 (m, 1H), 2.18 (dt, J = 13.9, 7.1 Hz, 1H), 2.09- 2.04 (m, 1H), 1.88 (dd, J = 9.8, 3.7 Hz, 1H), 1.70- 1.62 (m, 1H), 1.61-1.54 (m, 1H), 0.77 (t, J = 6.4 Hz, 6H). 473.9 3.1 1AV .sup.1H NMR (600 MHz, DMSO) 8.28 (d, J = 3.2 Hz, 3H), 8.25 (s, 1H), 4.08 (d, J = 7.0 Hz, 2H), 3.80 (dd, J = 12.3, 3.4 Hz, 1H), 3.56-3.50 (m, 1H), 3.35 (ddd, J = 14.2, 9.1, 4.9 Hz, 1H), 3.11 (dd, J = 12.4, 9.5 Hz, 1H), 3.06- 2.99 (m, 1H), 2.09-2.02 (m, 1H), 1.89 (dd, J = 9.0, 4.5 Hz, 1H), 1.72- 1.63 (m, 1H), 1.59 (dt, J = 10.8, 6.6 Hz, 1H), 1.30- 1.22 (m, 1H), 0.51-0.47 (m, 2H), 0.41- 0.36 (m, 2H). 473.9 3 1AW 0 .sup.1H NMR (600 MHz, DMSO) 8.25 (bs, 4H), 4.08 (d, J = 7.0 Hz, 2H), 3.80 (dd, J = 12.3, 3.4 Hz, 1H), 3.56- 3.50 (m, 1H), 3.35 (dd, J = 9.2, 4.3 Hz, 1H), 3.11 (dd, J = 12.4, 9.5 Hz, 1H), 3.06- 3.00 (m, 1H), 2.08-2.02 (m, 1H), 1.88 (dd, J = 9.1, 4.5 Hz, 1H), 1.67 (ddd, J = 13.7, 10.5, 5.5 Hz, 1H), 1.59 (dt, J = 11.0, 6.8 Hz, 1H), 1.30- 1.21 (m, 1H), 0.51-0.47 (m, 2H), 0.41- 0.36 (m, 2H). 461.8 2.9 1AX 1H NMR (600 MHz, DMSO) 8.91 (bs, 2H), 8.39 (s, 1H), 4.81 (hept, J = 7.0 Hz, 1H), 3.70- 3.62 (m, 1H), 3.31-3.17 (m, 4H), 3.05 (dd, J = 12.7, 8.5 Hz, 1H), 1.58 (d, J = 7.0 Hz, 3H), 1.51 (d, J = 6.9 Hz, 3H), 1.05 (d, J = 6.5 Hz, 1H). 447.8 2.7 1AY 1H NMR (600 MHz, DMSO) 8.43 (s, 1H), 8.09 (s, 1H), 4.78 (hept, J = 6.8 Hz, 1H), 3.92-3.82 (m, 1H), 3.70- 3.64 (m, 1H), 2.29 (td, J = 13.5, 7.2 Hz, 1H), 2.14- 2.04 (m, 1H), 1.55 (dd, J = 16.1, 6.9 Hz, 2H). 447.8 5.5 1AZ 1H NMR (300 MHz, dmso) 8.39 (s, 3H), 8.08 (s, 1H), 4.76 (dt, J = 13.8, 6.9 Hz, 1H), 3.83 (dd, J = 17.1, 6.9 Hz, 3H), 3.71- 3.58 (m, 2H), 2.25 (dt, J = 13.5, 6.6 Hz, 1H), 2.13- 1.98 (m, 1H), 1.53 (dd, J = 7.8, 7.1 Hz, 6H 447.9 2.9 1BA 1H NMR (600 MHz, DMSO) 7.98 (s, 1H), 4.83 (hept, J = 6.8 Hz, 1H), 4.57-4.51 (m, 1H), 3.45- 3.35 (m, 2H), 3.31-3.19 (m, 2H), 2.30- 2.18 (m, 1H), 2.08 (td, J = 13.2, 5.9 Hz, 1H), 1.50 (dd, J = 6.9, 2.0 Hz, 6H). 462.8 3.4 1BB 00 463.8 2.6 01 1BC 02 475.9 3.3 03 1BD 04 .sup.1H NMR (600 MHz, DMSO) 9.43 (bs, 1H), 9.12 (bs, 1H), 7.98 (s, 1H), 7.79 (d, J = 6.3 Hz, 1H), 4.81 (hept, J = 6.8 Hz, 1H), 4.56-4.49 (m, 1H), 3.44- 3.35 (m, 2H), 3.25 (tt, J = 13.1, 6.4 Hz, 2H), 2.28- 2.20 (m, 1H), 2.10-2.04 (m, 1H), 1.50 (dd, J = 6.8, 1.3 Hz, 6H). 447.8 2.9 05 1BF 06 1H NMR (600 MHz, DMSO) 9.47 (bs, 1H), 9.16 (bs, 1H), 7.98 (s, 1H), 7.82 (d, J = 6.4 Hz, 1H), 4.82 (hept, J = 6.9 Hz, 1H), 4.53 (dt, J = 11.4, 5.6 Hz, 1H), 3.45- 3.35 (m, 2H), 3.29-3.20 (m, 2H), 2.28- 2.21 (m, 1H), 2.08 (td, J = 13.3, 5.9 Hz, 1H), 1.50 (dd, J = 6.9, 1.7 Hz, 6H). 447.8 2.7 07 1BG 08 1H NMR (600 MHz, DMSO) 8.32 (s, 3H), 8.07 (s, 1H), 4.22-4.13 (m, 2H), 3.94- 3.88 (m, 3H), 3.83-3.76 (m, 2H), 2.33 (td, J = 13.7, 8.1 Hz, 1H), 2.13 (ddd, J = 12.3, 7.8, 4.1 Hz, 1H), 1.30-1.23 (m, 1H), 0.47 (d, J = 8.1 Hz, 2H), 0.40-0.37 (m, 2H). 459.9 2.9 09 1BH 0 1H NMR (600 MHz, DMSO) 8.02 (s, 1H), 7.51 (d, J = 5.8 Hz, 1H), 4.68- 4.64 (m, J = 13.8, 6.9 Hz, 1H), 4.63- 4.57 (m, J = 12.2, 6.1 Hz, 1H), 3.55 (dd, J = 12.0, 6.7 Hz, 1H), 3.41- 3.40 (m, 1H), 3.32-3.28 (m, 2H), 3.24 (dd, J = 11.9, 5.4 Hz, 1H), 2.31 (td, J = 14.8, 7.2 Hz, 1H), 2.10 (td, J = 13.4, 6.5 Hz, 1H), 1.49 (dd, J = 6.8, 2.3 Hz, 6H). 427.8 2.7 1BI .sup.1H NMR (600 MHz, DMSO) 8.54 (bs, 3H), 8.11 (s, 1H), 4.79 (hept, J = 6.9 Hz, 1H), 3.94- 3.90 (m, 3H), 3.79-3.70 (m, 2H), 2.35- 2.28 (m, 1H), 2.17-2.10 (m, 1H), 1.54 (dd, J = 19.0, 6.9 Hz, 6H). 427.8 2.6 1BJ .sup.1H NMR (600 MHz, DMSO) 8.30 (s, 1H), 8.06 (bs, 2H), 4.55 (hept, J = 7.0 Hz, 1H), 3.65 (dd, J = 12.3, 3.3 Hz, 1H), 3.50- 3.44 (m, 2H), 3.16 (dd, J = 12.4, 8.8 Hz, 1H), 3.09- 3.03 (m, 1H), 2.04 (dd, J = 13.0, 4.5 Hz, 1H), 1.91 (dd, J = 9.4, 4.5 Hz, 1H), 1.77- 1.70 (m, 1H), 1.64-1.56 (m, 1H), 1.53 (dd, J = 7.3, 1.3 Hz, 6H). 441.9 2.8 1BK .sup.1H NMR (600 MHz, DMSO) 9.25 (bs, 1H), 8.99 (bs, 1H), 8.01 (s, 1H), 7.72 (d, J = 6.2 Hz, 1H), 4.75 (hept, J = 6.9 Hz, 1H), 4.66-4.60 (m, 1H), 3.54- 3.47 (m, 2H), 3.29 (td, J = 11.9, 6.9 Hz, 2H), 2.30 (dt, J = 15.1, 7.1 Hz, 1H), 2.11 (dt, J = 13.3, 6.0 Hz, 1H), 1.49 (d, J = 6.9 Hz, 6H). 427.9 2.8 1BL 1H NMR (600 MHz, DMSO) 9.51 (bs, 2H), 8.32 (s, 1H), 4.61 (hept, J = 6.8 Hz, 1H), 3.54- 3.50 (m, 4H), 3.29 (bs, 4H), 1.53 (d, J = 6.9 Hz, 6H). 427.8 2.7 1BM 0 1H NMR (600 MHz, DMSO) 8.31 (s, 1H), 8.16 (s, 3H), 4.62 (hept, J = 6.9 Hz, 1H), 4.03-3.94 (m, 2H), 3.81 (td, J = 11.5, 2.4 Hz, 1H), 3.51 (d, J = 12.4 Hz, 1H), 3.39 (d, J = 11.3 Hz, 1H), 3.17- 3.07 (m, 2H), 3.01 (dd, J = 12.6, 10.4 Hz, 1H), 2.95- 2.88 (m, 1H), 1.56 (d, J = 6.9 Hz, 3H), 1.52 (d, J = 6.9 Hz, 457.9 2.7 3H). 1BN .sup.1H NMR (600 MHz, DMSO) 9.16 (bs, 1H), 9.12 (bs, 1H), 7.96 (s, 1H), 7.66 (t, J = 5.7 Hz, 1H), 4.70 (hept, J = 6.9 Hz, 1H), 4.03-3.97 (m, 2H), 3.61- 3.55 (m, 2H), 3.54-3.49 (m, 2H), 3.17 (d, J = 12.8 Hz, 1H), 3.04- 2.97 (m, 1H), 2.88-2.82 (m, 1H), 1.47 (d, J = 6.7 Hz, 6H). 457.9 2.8 1BS 463.8 2.3 1BT .sup.1H NMR (600 MHz, DMSO) 9.19 (bs, 1H), 8.87 (bs, 1H), 7.99 (s, 1H), 7.60 (d, J = 7.5 Hz, 1H), 4.81 (hept, J = 6.8 Hz, 1H), 4.31-4.24 (m, 1H), 3.43 (d, J = 12.5 Hz, 1H), 3.17 (d, J = 12.6 Hz, 1H), 2.95 (dd, J = 19.0, 9.5 Hz, 1H), 2.88 (dd, J = 19.1, 9.3 Hz, 1H), 2.01 (dd, J = 9.5, 3.7 Hz, 1H), 1.95 (dd, J = 9.5, 4.9 Hz, 1H), 1.78- 1.68 (m, 2H), 1.48 (dd, J = 6.8, 3.4 Hz, 6H). 441.8 2.8 1BV 441.9 2.7 1CA 0 1H NMR (300 MHz, dmso) 9.02 (bs, 1H), 8.79 (bs, 1H), 7.96 (s, 1H), 7.49 (d, J = 7.4 Hz, 1H), 4.76 (dt, J = 13.8, 6.9 Hz, 1H), 4.20-4.05 (m, 1H), 3.42- 3.31 (m, 1H), 3.19-3.07 (m, 1H), 2.93- 2.78 (m, 2H), 2.01-1.84 (m, 2H), 1.72- 1.59 (m, 2H), 1.47 (d, J = 6.8 Hz, 6H). 461.8 2.8 1CD 477.8 3.2 1CE 1H NMR (600 MHz, DMSO) 9.35 (bs, 1H), 9.27 (bs, 1H), 7.99 (s, 1H), 7.63 (d, J = 5.9 Hz, 1H), 4.82 (hept, J = 6.9 Hz, 1H), 4.42-4.40 (m, 1H), 4.29 (dt, J = 5.7, 3.0 Hz, 1H), 3.55 (td, J = 12.7, 6.5 Hz, 2H), 3.50- 3.46 (m, 2H), 1.50 (d, J = 6.9 Hz, 6H). 463.8 2.8 1CH 473.9 6.4 1CI 459.8 6.2 1CJ 0 459.8 2.9 1CK 459.8 2.7 1CL 387.9 2.5 1CM 1H NMR (600 MHz, DMSO) 8.43 (bs, 3H), 8.07 (s, 1H), 4.17 (t, J = 7.8 Hz, 2H), 3.93-3.86 (m, 3H), 3.81- 3.74 (m, 2H), 2.35-2.27 (m, 1H), 2.14 (dd, J = 8.9, 4.5 Hz, 1H), 1.80- 1.67 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). 447.9 2.6 1CN 343.9 2.5 1CO 0 343.9 2.5 1CP 1H NMR (300 MHz, dmso) 8.22 (s, 1H), 8.21-8.09 (m, 3H), 4.47 (dt, J = 13.8, 6.8 Hz, 1H), 3.51 (d, J = 12.8 Hz, 2H), 3.32- 3.16 (m, 1H), 3.12-2.99 (m, 2H), 2.05- 1.94 (m, 2H), 1.79-1.63 (m, 2H), 1.52 (d, J = 6.9 Hz, 6H). 461.9 2.6 1CQ 1H NMR (300 MHz, dmso) 8.02 (bs, 3H), 7.92 (s, 1H), 6.96 (d, J = 4.1 Hz, 1H), 4.87 (dd, J = 13.6, 6.8 Hz, 1H), 3.18-3.02 (m, 1H), 2.04- 1.91 (m, J = 9.8 Hz, 2H), 1.78-1.68 (m, 4H), 1.67- 1.55 (m, 2H), 1.47 (d, J = 6.8 Hz, 6H). 475.9 3.0 1CR 433.8 2.8 1CS 463.8 2.5 1CT 0 357.9 2.6 1CX 1H NMR (300 MHz, dmso) 8.51 (bs, 3H), 8.11 (s, 1H), 4.50 (dd, J = 9.1, 8.7 Hz, 2H), 4.39 (dt, J = 13.9, 6.9 Hz, 1H), 4.25 (d, J = 9.1 Hz, 2H), 4.19-4.08 (m, 1H), 1.50 (d, J = 6.9 Hz, 6H). 433.8 2.5 1CY 387.9 2.5 1DA 358.0 2.6 1DC 1H NMR (300 MHz, dmso) 8.37 (bs, 3H), 8.10 (s, 1H), 5.00-4.86 (m, 1H), 3.84- 3.72 (m, 3H), 3.62 (dd, J = 11.1, 7.5 Hz, 2H), 2.79- 2.60 (m, 2H), 2.44-2.32 (m, 2H), 2.30- 2.17 (m, 1H), 2.09-1.99 (m, 1H), 1.97- 1.85 (m, 1H), 1.83-1.70 (m, 1H). 459.8 2.6 1DF 0 463.8 2.2 1DG 1H NMR (600 MHz, DMSO) 9.34 (bs, 2H), 8.11 (s, 1H), 4.81 (p, J = 8.8 Hz, 1H), 3.45 (d, J = 4.8 Hz, 2H), 3.44 (s, 1H), 3.44- 3.42 (m, 1H), 3.25-3.19 (m, 4H), 2.61- 2.54 (m, 2H), 2.54-2.49 (m, 2H), 1.91- 1.84 (m, 1H), 1.84-1.75 (m, 1H). 459.8 2.8 1DJ 449.8 2.2 1DK 1H NMR (600 MHz, DMSO) 9.08-8.99 (m, 1H), 8.87- 8.80 (m, 1H), 7.91 (s, 1H), 7.48 (d, J = 7.2 Hz, 1H), 4.78 (sept., J = 6.8 Hz, 1H), 3.31- 3.24 (m, 2H), 3.04-2.96 (m, 2H), 2.07- 2.00 (m, 2H), 1.85-1.76 (m, 2H), 1.46 (d, J = 6.9 Hz, 6H). 461.8 2.8 1DN 477.9 5.4 1DO 0 1H NMR (300 MHz, dmso) 8.05 (bs, 3H), 7.90 (s, 1H), 7.09 (d, J = 1.8 Hz, 1H), 4.22 (q, J = 6.9 Hz, 2H), 3.10 (s, 1H), 2.05- 1.92 (m, 2H), 1.81-1.69 (m, 4H), 1.69- 1.59 (m, 2H), 1.17 (t, J = 7.0 Hz, 3H). 461.9 2.8 1DP 1H NMR (300 MHz, dmso) 8.97 (bs, 2H), 8.33 (s, 1H), 4.17 (dt, J = 12.3, 7.3 Hz, 2H), 3.95- 3.86 (m, 1H), 3.42 (dd, J = 10.6, 4.9 Hz, 2H), 3.36 (d, J = 3.6 Hz, 1H), 3.26 (t, J = 5.1 Hz, 2H), 3.11 (dd, J = 12.8, 6.4 Hz, 1H), 1.27 (t, J = 7.1 Hz, 3H), 1.16 (d, J = 6.7 Hz, 3H). 427.9 2.3 1DQ 1H NMR (300 MHz, dmso) 8.24 (bs, 3H), 8.19 (s, 1H), 4.13 (q, J = 7.3 Hz, 2H), 3.53 (d, J = 13.1 Hz, 1H), 3.44- 3.31 (m, 1H), 3.24-3.04 (m, 3H), 2.08- 1.97 (m, 1H), 1.95-1.84 (m, 1H), 1.65 (dd, J = 21.5, 13.9 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H). 427.8 2.5 1DR 1H NMR (300 MHz, dmso) 8.38 (bs, 3H), 8.03 (s, 1H), 4.24 (q, J = 7.3 Hz, 4H), 3.99- 3.89 (m, 3H), 3.87-3.77 (m, 2H), 2.29 (dd, J = 13.6, 7.8 Hz, 1H), 2.15 (d, J = 4.5 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H). 413.8 2.4 1DS 427.9 4.9 1DT 0 441.9 5.6 1DU 1H NMR (600 MHz, DMSO) 7.88 (s, 1H), 7.85-7.79 (m, 3H), 7.28 (d, J = 7.8 Hz, 1H), 4.09 (q, J = 7.1 Hz, 2H), 3.74- 3.66 (m, 1H), 3.05-2.97 (m, 1H), 2.04- 2.00 (m, 2H), 2.00-1.95 (m, 2H), 1.48- 1.37 (m, 4H), 1.16 (t, J = 7.1 Hz, 3H). 461.9 2.3 1DV 441.9 2.3 1DW 1DY 1DZ 00 01 1EA 02 03 1EB 04 05

3.2. Compounds of Example 2

(63) ##STR00206##

5,6-dibromo-4-nitro-2-(piperazin-1-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole hydrochloride (Example 2A)

(64) Tert-butyl 4-(5,6-dibromo-4-nitro-1H-1,3-benzodiazol-2-yl)piperazine-1-carboxylate (Method 4A)(0.4 mmol, 200 mg) was dissolved in acetonitrile (5 ml). Next, NaOH (0.5 mmol, 19 mg) was added. The mixture was stirred at RT for 0.5 h. Then 2-Iodopropane (32 mmol, 538 mg) was added dropwise. The resulting mixture was stirred at 85 C. in a sealed tube until the reaction was complete (18 hours) by LC/MS. The mixture was allowed to cool to RT and concentrated in-vacuo. The product was taken up into ethyl acetate and washed with water. The organic extract was dried over MgSO4, filtered and concentrated. The product was purified on silica gel using EA/hex (1:4). The obtained product (0.3 mmol, 180 mg) was dissolved in MeOH (3 ml), then hydrogen chloride, (4M in 1,4-dioxane, 1 ml) was added dropwise. The resulting mixture was stirred at RT overnight. Solid was filtered and washed with Et.sub.2O to afford 5,6-dibromo-4-nitro-2-(piperazin-1-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole hydrochloride (130 mg). .sup.1H NMR (600 MHz, DMSO) 9.37 (s, 2H), 8.28 (s, 1H), 4.62 (hept, J=6.9 Hz, 1H), 3.47-3.44 (m, 4H), 3.28-3.26 (m, 4H), 1.54 (d, J=6.9 Hz, 6H); m/z 472; rt 2.4.

(65) The following compounds were prepared by the procedure of Example 2A, using the appropriate starting materials.

(66) TABLE-US-00002 1HNMR Ex. Compound (400 MHz) m/z RT SM 2B 5,6-dibromo-4-nitro- 2-(piperazin-1-yl)-1- (prop-2-yn-1-yl)-1H- 1,3-benzodiazole hydrochloride 07 1H NMR (600 MHz, DMSO) 9.52 (s, 2H), 8.17 (s, 1H), 5.10 (d, J = 2.4 Hz, 2H), 3.65-3.62 (m, 4H), 3.61 (t, J = 2.4 Hz, 1H), 3.28 (s, 4H). 443.9 2.5 tert-butyl 4-(5,6- dibromo-4-nitro-1H-1,3- benzodiazol-2- yl)piperazine-1- carboxylate Method 4A 08 and propargyl bromide (commercial) 2C 5,6-dibromo-4-nitro- 2-(piperazin-1-yl)-1- [2-(piperazin-1- yl)ethyl]-1H-1,3- benzodiazole hydrochloride 09 1H NMR (600 MHz, DMSO) 9.92 (bs, 2H), 9.44 (bs, 2H), 8.45 (s, 1H), 4.68- 4.56(m, 2H), 3.66- 3.55 (m, 8H), 3.56- 3.49 (m, 8H). 473.9 3.0 tert-butyl 4-(5,6- dibromo-4-nitro-1H-1,3- benzodiazol-2- yl)piperazine-1- carboxylate Method 4A 0 and tert-butyl 4-(2- chloroethyl)piperazine- 1- Carboxylate (commercial) 2D 5,6-dibromo-1- cyclopentyl-4-nitro- 2-(piperazin-1-yl)- 1H-1,3-benzodiazole hydrochloride .sup.1H NMR (600 MHz, DMSO) 9.41 (bs, 2H), 7.99 (s, 1H), 4.72 (p, J = 9.0 Hz, 1H), 3.51-3.42 (m, 4H), 3.31-3.22 (m, 4H), 2.12-2.00 (m, 4H), 1.99-1.91 (m, 2H), 1.73 (dt, J = 11.2, 4.6 Hz, 2H). 473.9 6.6 tert-butyl 4-(5,6- dibromo-4-nitro-1H-1,3- benzodiazol-2- yl)piperazine-1- carboxylate Method 4A and cyclopentyl iodide (commercial) 2E 5,6-dibromo-1-(3- methylbut-2-en-1- yl)-4-nitro-2- (piperazin-1-yl)-1H- 1,3-benzodiazole hydrochloride 473.9 3.3 tert-butyl 4-(5,6- dibromo-4-nitro-1H-1,3- benzodiazol-2- yl)piperazine-1- carboxylate Method 4A and 1-bromo-3-methyl- but-2-en (commercial) 2F 5,6-dibromo-1- (cyclobutylmethyl)- 4-nitro-2-(piperazin- 1-yl)-1H-1,3- benzodiazole hydrochloride 1H NMR (600 MHz, DMSO) 8.20 (s, 1H), 4.21 (d, J = 7.4 Hz, 2H), 3.26 (dd, J = 5.9, 3.9 Hz, 4H), 2.89-2.85 (m, 4H), 2.79-2.70 (m, 1H), 1.91-1.77 (m, 4H), 1.75-1.68 (m, 2H). 459.9 2.9 tert-butyl 4-(5,6- dibromo-4-nitro-1H-1,3- benzodiazol-2- yl)piperazine-1- carboxylate Method 4A and methylcyclobutyl bromide 2G N-(3-aminopropyl)- 5,6-dibromo-4-nitro- 1-(prop-2-yn-1-yl)- 1H-1,3-benzodiazol- 2-amine hydrochloride 461.9 3.2 tert-butyl N-{3-[(5,6- dibromo-4-nitro-1H-1,3- benzodiazol-2- yl)amino]propyl}carbamate Method 4C and propargyl bromide 2H N-(3-aminopropyl)- 5,6-dibromo-1- cyclopentyl-4-nitro- 1H-1,3-benzodiazol- 2-amine hydrochloride 463 2.3 tert-butyl N-{3-[(5,6- dibromo-4-nitro-1H-1,3- benzodiazol-2- yl)amino]propyl}carbamate 0 Method 4C And cyclopentyl iodide 2I 5,6-dibromo-1- (butan-2-yl)-4-nitro- 2-(piperazin-1-yl)- 1H-1,3-benzodiazole hydrochloride 486.9 3.4 tert-butyl 4-(5,6- dibromo-4-nitro-1H-1,3- benzodiazol-2- yl)piperazine-1- carboxylate Method 4A and 2-butyl bromide 2J 3-[5,6-dibromo-4- nitro-2-(piperazin-1- yl)-1H-1,3- benzodiazol-1- yl]propan-1- aminehydrochloride 462.9 1.9 tert-butyl 4-(5,6- dibromo-4-nitro-1H-1,3- benzodiazol-2- yl)piperazine-1- carboxylate Method 4A and tert-butyl N-(3- bromopropyl)carbamate 2L 2-[5,6-dibromo-4- nitro-2-(piperazin-1- yl)-1H-1,3- benzodiazol-1- yl]acetamide tert-butyl 4-(5,6- dibromo-4-nitro-1H-1,3- benzodiazol-2- yl)piperazine-1- carboxylate Method 4A and 2-bromoacetamide

3.3. Compounds of Example 3

(67) ##STR00227##

(3R)-1-[1-(3-aminopropyl)-5,6-dibromo-4-nitro-1H-1,3-benzodiazol-2-yl]pyrrolidin-3-amine (Example 3A)

(68) A suspension of 3-{2-[(3R)-3-aminopyrrolidin-1-yl]-5,6-dibromo-4-nitro-1H-1,3-benzodiazol-1-yl}propan-1-ol (53.2 mg, 0.115 mmol) in MeOH/triethylamine (7:1 v/v, 3.1 mL) was stirred at 0 C. for 10 min. Di-tert-butyl dicarbonate (67.8 mg, 0.264 mmol) in MeOH (1.3 mL) was added slowly over 10 min under an argon atmosphere. The mixture was stirred at 0 C. for 1 h and then at room temperature for 16 h to completion (checked by TLC, AcOEt-Hex: 4-1). The solvent was removed under reduced pressure. The solid obtained was dissolved in CH2Cl2 (4 mL) and the resulting solution was washed with water (3 mL3). The organic layer was separated, dried over anhydrous Na2SO4 and evaporated to give tert-butyl N-[(3R)-1-[5,6-dibromo-1-(3-hydroxypropyl)-4-nitro-1H-1,3-benzodiazol-2-yl]pyrrolidin-3-yl]carbamate as a yellow solid (62.9 mg, 0.108 mmol, 97%). It was dissolved without any further purification in dry tetrahydrofuran (1.1 mL) with triphenylphosphine (32.3 mg, 2.0 mmol) and phtalimide (58.0 mg, 2.0 mmol. A solution of diisopropyl azodicarboxylate (48 uL, 2.2 mmol) in tetrahydrofuran (0.4 mL) was added dropwise with stirring overnight at room temperature. Thus, the solvent was removed by evaporation and the residue taken into CH2Cl2 (4 mL), washed with a solution of sodium bicarbonate and water, dried over magnesium sulfate, filtered ad evaporated to dryness. The residue was chromatographed on a silica gel column eluted with ethyl acetate-hexane mixture (4:1) to obtain tert-butyl N-[(3R)-1-{5,6-dibromo-1-[3-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)propyl]-4-nitro-1H-1,3-benzodiazol-2-yl}pyrrolidin-3-yl]carbamate (72.6 mg, 0.102 mmol, 99%). m/z=693.0, rt=3.7 min. It was suspended in absolute ethanol (3.0 mL) and a solution of monohydrate hydrazine (50.4 L, 1.017 mmol) in absolute ethanol (1.0 mL) was slowly added. The mixture was refluxed for 2.0 h. evaporation of the solvent gave solid which was dissolved 4.4 M HCl in ethanol (4.0 mL) is added. The mixture is stirred at room temperature until the reaction is complete (18 h) by LCMS. Diethyl ether (5.0 ml) is added, product is filtered off, washed with diethyl ether, dried and purified by preparative HPLC to afford (3R)-1-[1-(3-aminopropyl)-5,6-dibromo-4-nitro-1H-1,3-benzodiazol-2-yl]pyrrolidin-3-amine. m/z 462.9; rt 1.8 min.

(69) The following compound was prepared by the procedure of Example 3A, using the appropriate starting materials.

(70) TABLE-US-00003 1HNMR Ex. Compound (400 MHz) m/z rt SM 3B (3S)-1-[1-(2- aminoethyl)-5,6- dibromo-4-nitro-1H- 1,3-benzodiazol-2- yl]pyrrolidin-3- amine 2-{2-[(3S)-3- aminopyrrolidin-1- yl]-5,6-dibromo-4- nitro-1H-1,3- benzodiazol-1- yl}ethan-1-ol

3.4. Compounds of Example 4

(71) ##STR00230##

5-methyl-6-bromo-4-nitro-2-(piperazin-1-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole (Example 4A) and 5-bromo-6-methyl-4-nitro-2-(piperazin-1-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole (Example 4B)

(72) 5,6-dibromo-4-nitro-2-(piperazin-1-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole (Example 2A) (0.08 mmol, 50 mg) was suspended in a mixture of 1,4-dioxane/H2O (10:1) (1.5 ml). methyl boronic acid (0.2 mmol, 39.5 mg) and Cs2CO3 (0.16 mmol, 34.5 mg) were added. The reaction mixture was flushed with argon for 5 min. Then [1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) was added. The resulting mixture was stirred at 130 C. until the reaction was complete (16 hours) by LC/MS. The mixture was allowed to cool to RT and filtered through Celite. Solvent was evaporated in-vacuo. The product was dissolved in ethyl acetate and washed with water. The organic extract was dried over MgSO4, filtered and concentrated.

(73) The products were purified on HPLC to afford 5-bromo-6-methyl-4-nitro-2-(piperazin-1-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole trifluoroacetate (10 mg). m/z 383.9; rt 2.5; 5-methyl-6-bromo-4-nitro-2-(piperazin-1-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole (5 mg) m/z 383.9, rt 2.6; 1H NMR (300 MHz, dmso) 9.19 (bs, 2H), 8.13 (s, 1H), 4.59 (sept, J=6.9 Hz, 1H), 3.42-3.35 (m, 4H), 3.31-3.21 (m, 4H), 2.34 (s, 3H), 1.51 (d, J=6.9 Hz, 6H).

3.5. Compounds of Example 8

(74) ##STR00231##

5,6-dibromo-1-ethyl-2-(piperazin-1-yl)-1H-1,3-benzodiazole-4-carbonitrilehydrochloride (Example 8A)

(75) tert-butyl 4-(5,6-dibromo-4-cyano-1-ethyl-1H-1,3-benzodiazol-2-yl)piperazine-1-carboxylate (Method 8A) (900 mg, 2.18 mmol) was dissolved in 1,4-dioxane (5.0 ml) and 4M HCl in dioxane (2.0 ml) was added. The mixture was stirred at room temperature until the reaction was complete (18 hrs) by LC/MS. Diethyl ether (10.0 ml) was added, product was filtered off, washed with diethyl ether and dried to afford 5,6-dibromo-1-ethyl-2-(piperazin-1-yl)-1H-1,3-benzodiazole-4-carbonitrile hydrochloride (820 mg, 1.8 mmol) 1H NMR (600 MHz, DMSO) 9.47 (s, 2H), 8.24 (s, 1H), 4.17 (q, J=7.2 Hz, 2H), 3.67-3.62 (m, 4H), 3.29 (s, 4H), 1.32 (t, J=7.2 Hz, 3H), m/z 413.9; rt 2.2.

(76) The following example was prepared by the procedure of Examples 8A, using the appropriate starting materials:

(77) TABLE-US-00004 1HNMR Ex. Compound (400 MHz) m/z RT SM 8G 2-[(3R)-3- aminopyrrolidin-1- yl]-5,6-dibromo-1- [(3S)-pyrrolidin-3- yl]-1H-1,3- benzodiazole-4- carbonitrile 454.9 1.6 tert-butyl (3S)-3-{5,6- dibromo-2-[(3R)-3- {[(tert- butoxy)carbonyl]amino} pyrrolidin-1-yl]-4- cyano-1H-1,3- benzodiazol-1- yl}pyrrolidine-1- carboxylate

3.6. Compounds of Example 9

(78) ##STR00234##

5,6-dibromo-1-ethyl-2-(piperazin-1-yl)-4-(trifluoromethyl)-1H-1,3-benzodiazolehydrochloride (Example 9A)

(79) tert-butyl 4-(5,6-dibromo-1-ethyl-4-iodo-1H-1,3-benzodiazol-2-yl)piperazine-1-carboxylate (150 mg, 0.24 mmol) methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (0.092 ml, 0.73 mmol) and copper (I) iodide (4.7 mg, 0.024 mmol) were dissolved in DMF (3.0 ml). The resulting mixture was stirred at 150 C. under microwave conditions until the reaction was complete (10 min) by LC/MS. The mixture was allowed to cool to RT and concentrated in-vacuo. The residue was dissolved in ethyl acetate and washed with water. The organic extract was dried over MgSO4, filtered and concentrated. The product was purified on silica gel using EA/hex (1:1). The product was dissolved in 1,4-dioxane (1.0 ml) and 4M HCl in dioxane (1.0 ml) was added. The mixture was stirred at room temperature until the reaction was complete (18 hrs) by LC/MS. The mixture was concentrated in-vacuo and purified on HPLC to afford 5,6-dibromo-1-ethyl-2-(piperazin-1-yl)-4-(trifluoromethyl)-1H-1,3-benzodiazole hydrochloride (22 mg, 0.05 mmol). 1H NMR (600 MHz, DMSO) 9.21 (bs, 2H), 8.23 (s, 1H), 4.16 (t, J=7.2 Hz, 2H), 3.59-3.55 (m, 4H), 3.29 (bs, 4H), 1.31 (t, J=7.1 Hz, 3H); m/z 456.8; rt 3.1.

(80) The following example was prepared by the procedure of Example 9A, using the appropriate starting materials:

(81) TABLE-US-00005 1HNMR Ex. Compound (400 MHz) m/z RT SM 9B 6-bromo-4-nitro-2- (piperazin-1-yl)-1- (propan-2-yl)-5- (trifluoromethyl)- 1H-1,3- benzodiazole 1H NMR (300 MHz, dmso) 8.94 (s, 2H), 8.29 (s, 1H), 4.21 (q, J = 7.1 Hz, 2H), 3.58- 3.45 (m, 4H), 3.27 (s, 4H), 1.33(t, J = 7.1 Hz, 3H). 421.9 2.7 tert-butyl N-[1-(4- amino-5,6-dibromo-1- ethyl-1H-1,3- benzodiazol-2- yl)piperidin-3- yl]carbamate: Method 12A

3.7. Compounds of Example 21

(82) ##STR00237##

5,6-dibromo-1-cyclopropyl-4-nitro-2-(piperazin-1-yl)-1H-1,3-benzodiazole trifluoroacetate (Example 21A)

(83) tert-butyl 4-(5,6-dibromo-1H-1,3-benzodiazol-2-yl)piperazine-1-carboxylate (150 mg, 0.33 mmol) was dissolved in dichloroethane (5.0 ml). Cyclopropylboronic acid (56 mg, 0.65 mmol), copper (II) acetate (59 mg, 0.33 mol), 2,2-bipyridine (51 mg, 0.65 mmols) and sodium carbonate (70 mg, 0.65 mmol) were added. The resulting mixture was stirred at 60 C. until the reaction was complete (3 days) by LC/MS. The mixture was allowed to cool to RT and concentrated in-vacuo. The product was taken up into DMC and washed with water. The organic extract was dried over MgSO4, filtered and concentrated. The product was purified on silica gel using EA/hex (1:4). The product was dissolved in sulfuric acid (conc.) (2.0 ml) and stirred 0 C. for 30 min, then potassium nitrate (12 mg, 0.12 mmol) was added in one portion and stirred at 0 C. for additional 3 hrs. The reaction mixture was left to warm to room temperature and was stirred until the reaction was complete (16 hrs). The mixture was poured onto ice. The product was taken up into DCM, dried over MgSO.sub.4, filtered and concentrated. The product was purified on HPLC to afford 5,6-dibromo-1-cyclopropyl-4-nitro-2-(piperazin-1-yl)-1H-1,3-benzodiazole trifluoroacetate (3.2 mg, 0.007 mmol); m/z 455.9; rt 3 min.

3.8. Compounds of Example 22

(84) ##STR00238##

4-(5,6-dibromo-1-ethyl-4-nitro-1H-1,3-benzodiazol-2-yl)piperazin-2-one (Example 22A)

(85) 2,5,6-tribromo-1-ethyl-4-nitro-1H-1,3-benzodiazole (100 mg, 0.23 mmol) and 2-piperazinone (117 mg, 1.17 mmol) were dissolved in EtOH (3.0 ml). The resulting mixture was stirred at temperature 170 C. under microwave conditions until the reaction was complete (20 min) by LC/MS. The mixture was allowed to cool to RT and concentrated in-vacuo. The product was filtered off, washed with EtOH and dried to afford-(5,6-dibromo-1-ethyl-4-nitro-1H-1,3-benzodiazol-2-yl)piperazin-2-one (97 mg, 0.22 mmol); m/z 462.9; rt 3.1 min.

(86) The following examples were prepared by the procedure of Example 22A, using the appropriate starting materials:

(87) TABLE-US-00006 1HNMR Ex. Compound (400 MHz) m/z RT SM 22B 4-[(5,6-dibromo-1- ethyl-4-nitro-1H-1,3- benzodiazol-2- yl)amino]cyclohexan- 1-ol 1H NMR (600 MHz, DMSO) 7.85 (s, 1H), 7.16 (d, J = 7.8 Hz, 1H), 4.56 (d, J = 3.3 Hz, 1H), 4.08 (q, J = 7.1 Hz, 2H), 3.68 (dtd, J = 15.2, 7.7, 4.0 Hz, 1H), 3.40 (ddd, J = 21.1, 13.6, 7.2 Hz, 1H), 1.92 (d, J = 11.4 Hz, 2H), 1.85 (d, J = 10.9 Hz, 2H), 1.44-1.34 (m, 2H), 1.29-1.21 (m, 2H), 1.15 (t, J = 7.1 Hz, 3H). 447.9 2.6 2,5,6-tribromo-1- ethyl-4-nitro-1H-1,3- benzodiazole 0 Method 3A And4- aminocyclohexan-1- ol 22F (3S)-1-(5,6-dibromo- 1-ethyl-4-nitro-1H- 1,3-benzodiazol-2- yl)pyrrolidin-3-ol 1H NMR (600 MHz, DMSO) 7.96 (s, 1H), 5.05 (d, J = 3.6 Hz, 1H), 4.38 (t, J = 6.1 Hz, 1H), 4.32-4.19 (m, 2H), 3.80- 3.75 (m, 2H), 3.72- 3.67 (m, 1H), 3.50 (d, J = 10.6 Hz, 1H), 2.00 (dtd, J = 12.9, 8.8, 4.4 Hz, 1H), 1.91- 1.86 (m, 1H), 1.27 (t, J = 7.1 Hz, 3H). 379.8 2.2 2,5,6-tribromo-1- ethyl-4-nitro-1H-1,3- benzodiazole Method 3A And(3S)-pyrrolidin- 3-ol 22G 1-[5,6-dibromo-4- nitro-1-(propan-2- yl)-1H-1,3- benzodiazol-2- yl]piperidin-3- .sup.1H NMR (600 MHz, DMSO) 8.18 (s, 1H), 4.93 (d, J = 4.5 Hz, 1H), 4.55 (hept, J = 6.9 Hz, 1H), 3.74- 3.68 (m, 1H), 3.42 (dd, J = 12.2, 3.6 Hz, = 1H), 3.28 (dd, J = 11.2, 4.2 Hz, 1H), 3.02 (ddd, J = 12.5, 10.0, 2.8 Hz, 1H), 2.84 (dd, J = 12.2, 8.4 Hz, 1H), 1.90-1.85 (m, 1H), 1.82 (dd, J = 9.1, 4.2 Hz, 1H), 1.64-1.55 (m, 1H), 1.53 (dd, J = 7.3, 1.2 Hz, 6H), 1.44-1.34 (m, 1H). 476.9 3.5 2,5,6-tribromo-1-(2- propyl)-4-nitro-1H- 1,3- benzodiazole Method 3B And piperidin-3-ol 22H {1-[5,6-dibromo-4- nitro-1-(propan-2- yl)-1H-1,3- benzodiazol-2- yl]piperidin-3- yl}methanol 1H NMR (600 MHz, DMSO) 8.19 (s, 1H), 4.56- 4.49 (m, 1H), 3.54- 3.50 (m, 1H), 3.40-3.36 (m, 2H), 3.29 (dd, J = 10.6, 8.1 Hz, 1H), 2.97-2.90 (m, 1H), 2.77 (dd, J = 12.4, 10.0 Hz, 1H), 1.83-1.61 (m, 4H), 1.54 (dd, J = 20.4, 6.9 Hz, 6H), 1.21-1.13 (m, 1H). 428.9 3 2,5,6-tribromo-1-(2- propyl)-4-nitro-1H- 1,3- benzodiazole Method 3B And piperidin-3-yl- methanol 22I (3S)-1-[5,6-dibromo- 4-nitro-1-(propan-2- yl)-1H-1,3- benzodiazol-2- yl]piperidin-3- ol 465.9 2.8 2,5,6-tribromo-1-(2- propyl)-4-nitro-1H- 1,3- benzodiazole Method 3B (3S)-piperidin-3-ol 22J N-[2-(2- aminoethoxy)ethyl]- 5,6-dibromo-4-nitro- 1-(propan-2-yl)-1H- 1,3-benzodiazol-2- amine .sup.1H NMR (600 MHz, DMSO) 8.04 (bs, 2H), 7.90 (s, 1H), 7.63 (t, J = 5.4 Hz, 1H), 4.84 (hept, J = 6.8 Hz, (m, 4H), 3.56 (q, J = 5.6 Hz, 2H), 2.97 (bs, 2H), 1.48 (d, J = 6.9 Hz, 6H). 448.8 3.8 2,5,6-tribromo-1-(2- propyl)-4-nitro-1H- 1,3- benzodiazole 0 Method 3B And 2-(2- aminoethoxy)ethanamine 22K 5,6-dibromo-2- (morpholin-4-yl)-4- nitro-1-(propan-2- yl)-1H-1,3- benzodiazole 1H NMR (600 MHz, DMSO) 8.23 (s, 1H), 4.61 (hept, J = 6.9 Hz, 1H), 3.78-3.74 (m, 5H), 3.23- 3.20 (m, 4H), 1.54 (d, J = 6.9 Hz, 6H). 427.9 5.8 2,5,6-tribromo-1-(2- propyl)-4-nitro-1H- 1,3- benzodiazole Method 3B And morpholine 22L 1-N-(5,6-dibromo-1- ethyl-4-nitro-1H-1,3- benzodiazol-2- yl)cyclohexane-1,2- diamine 461.9 2.9 2,5,6-tribromo-1- ethyl-4-nitro-1H-1,3- benzodiazole Method 3A And 1,2- cyclohexanediamine 22M 5,6-dibromo-1-ethyl- 2-(4-methyl-1,4- diazepan-1-yl)-4- nitro-1H-1,3- benzodiazole .sup.1H NMR (600 MHz, DMSO) 8.01 (s, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.69-3.64 (m, 4H), 2.70 (dd, J = 6.2, 3.4 Hz, 2H), 2.56-2.54 (m, 2H), 2.28 (s, 3H), 1.95-1.90 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H). 461.9 2.5 2,5,6-tribromo-1- ethyl-4-nitro-1H-1,3- benzodiazole Method 3A And N- methylhomopiperazine 22N 1-N-(5,6-dibromo-1- ethyl-4-nitro-1H-1,3- benzodiazol-2- yl)benzene-1,2- diamine 455.8 3.2 2,5,6-tribromo-1- ethyl-4-nitro-1H-1,3- benzodiazole Method 3A And ortho- phenylenediamine 22O 5,6-dibromo-N-({1- [2- (dimethylamino)ethyl] pyrrolidin-3- yl}methyl)-1-ethyl- 4-nitro-1H-1,3- benzodiazol-2- amine 519 2.3 2,5,6-tribromo-1- ethyl-4-nitro-1H-1,3- benzodiazole 0 Method 3A And {1-[2- (dimethylamino)ethyl] pyrrolidin-3- yl}methanamine 22P 5,6-dibromo-1-ethyl- 4-nitro-N-[3- (pyrrolidin-1- yl)propyl]-1H-1,3- benzodiazol-2- amine 1H NMR (600 MHz, DMSO) 7.87 (s, 1H), 7.59 (t, J = 5.3 Hz, 1H), 4.07 (q, J = 7.1 Hz, 2H), 3.41 (dd, J = 12.3, 6.8 Hz, 2H), 2.54 (d, J = 12.3 Hz, 6H), 1.81- 1.77 (m, 2H), 1.71 (bs, 4H), 1.18 (t, J = 7.1 Hz, 3H). 475.9 2.7 2,5,6-tribromo-1- ethyl-4-nitro-1H-1,3- benzodiazole Method 3A 3-(pyrrolidin-1- yl)propan-1-amine 22R 5,6-dibromo-1-ethyl- 4-nitro-2-{4{3- (piperazin-1- yl)propyl]piperazin- 1-yl}-1H-1,3- benzodiazole 1H NMR (600 MHz, DMSO) 9.83 (bs, 2H), 8.23 (s, 1H), 4.20 (q, J = 7.1 Hz, 2H), 3.86- 3.71 (m, 16H), 3.33- 3.26 (m, 4H), 2.29-2.22 (m, 2H), 1.33 (t, J = 7.2 Hz, 3H). 560 2.2 2,5,6-tribromo-1- ethyl-4-nitro-1H-1,3- benzodiazole Method 3A And1-[3-(piperazin- 1- yl)propyl]piperazine 22V 1-N-(5,6-dibromo-1- ethyl-4-nitro-1H-1,3- benzodiazol-2- yl)benzene-1,3- diamine 1H NMR (600 MHz, DMSO) 9.10 (s, 1H), 8.03 (s, 1H), 7.03-6.95 (m, 2H), 6.86 (s, 1H), 6.29 (d, J = 7.4 Hz, 1H), 5.07 (s, 2H), 4.31 (q, J = 7.0 Hz, 2H), 1.24 (t, J = 7.1 Hz, 3H). 462 2.5 2,5,6-tribromo-1- ethyl-4-nitro-1H-1,3- benzodiazole Method 3A And meta- phenylenediamine 22W 5,6-dibromo-2-(3,3- dimethylpiperazin-1- yl)-1-ethyl-4-nitro- 1H-1,3- benzodiazole 1H NMR (600 MHz, DMSO) 8.25 (s, 1H), 4.21 (q, J = 7.1 Hz, 2H), 3.46 (d, J = 5.2 Hz, 2H), 3.40-3.25 (m, 6H), 1.39 (s, 6H), 1.32 (t, J = 7.2 Hz, 3H). 447.9 2.5 2,5,6-tribromo-1- ethyl-4-nitro-1H-1,3- benzodiazole Method 3A And 2,2- dimethylpiperazine 22X 5,6-dibromo-1-ethyl- 2-(3- methylpiperazin-1- yl)-4-nitro-1H-1,3- benzodiazole .sup.1H NMR (600 MHz, DMSO) 9.35 (bs, 2H), 8.22 (s, 1H), 4.19 (q, J = 7.3 Hz, 2H), 3.71 (d, J = 13.3 Hz, 2H), 3.46 (ddd, J = 9.9, 6.6, 3.2 Hz, 1H), 3.37 (dd, J = 22.3, 9.7 Hz, 2H), 3.26-3.14 (m, 2H), 1.32 (t, J = 7.2 Hz, 3H), 1.29 (d, J = 6.5 Hz, 3H). 433.9 1.6 2,5,6-tribromo-1- ethyl-4-nitro-1H-1,3- benzodiazole 0 Method 3A And 2- methylpiperazine 22Z 5,6-dibromo-2-[(3S)- 3-methylpiperazin-1- yl]-4-nitro-1- (propan-2-yl)-1H- 1,3- benzodiazole 1H NMR (600 MHz, DMSO) 8.20 (s, 1H), 4.54 (dq, J = 13.7, 6.9 Hz, 1H), 3.30- 3.26 (m, 2H), 2.88 (tdd, J = 13.5, 10.0, 7.2 Hz, 4H), 2.58 (dd, J = 11.8, 10.3 Hz, 1H), 1.54 (d, J = 6.9 Hz, 3H), 1.52 (d, J = 6.9 Hz, 3H), 0.99 (d, J = 6.3 Hz, 3H). 477.9 2.8 2,5,6-tribromo-1-(2- propyl)-4-nitro-1H- 1,3- benzodiazole Method 3B and (2S)-2- methylpiperazine 22AA 5,6-dibromo-2- [(3R)-3- methylpiperazin-1- yl]-4-nitro-1- (propan-2-yl)-1H- 1,3- benzodiazole 461.8 3 2,5,6-tribromo-1-(2- propyl)-4-nitro-1H- 1,3- benzodiazole Method 3B And (2R)-2- methylpiperazine 22AB N-(3-amino-2- methoxypropyl)-5,6- dibromo-4-nitro-1- (propan-2-yl)-1H- 1,3-benzodiazol-2- amine 465.8 2.7 2,5,6-tribromo-1-(2- propyl)-4-nitro-1H- 1,3- benzodiazole Method 3B And 2-methoxy-1,3- diaminopropane 22AC 2-[(3-amino-2- methoxypropyl)amino]- 5,6-dibromo-1- (propan-2-yl)-1H- 1,3-benzodiazole-4- carbonitrile 445.9 2.3 2,5,6-tribromo-1- (propan-2-yl)-1H- 1,3-benzodiazole-4- carbonitrile Method 8B And 2-methoxy-1,3- propylenediamine 22AD {4-[5,6-dibromo-4- nitro-1-(propan-2- yl)-1H-1,3- benzodiazol-2- yl]piperazin-2- yl}methanol 1H NMR (600 MHz, DMSO) 9.19 (bs, 1H), 8.78 (bs, 1H), 8.30 (s, 1H), 5.52 (bs, 1H), 4.63 (hept, J = 7.0 Hz, 1H), 3.68 (dd, J = 11.7, 4.5 Hz, 1H), 3.61 (dd, J = 11.6, 5.6 Hz, 1H), 3.58-3.51 (m, 2H), 3.51-3.44 (m, 2H), 3.26 (ddd, J = 14.1, 10.0, 4.2 Hz, 2H), 3.20 (dd, J = 13.6, 10.7 Hz, 1H), 1.54 (dd, J = 26.9, 6.9 Hz, 6H). 477.9 2.6 2,5,6-tribromo-1-(2- propyl)-4-nitro-1H- 1,3- benzodiazole 0 Method 3B And piperazin-2- ylmethanol 22AE 5,6-dibromo-2- [(1R,4R)-2,5- diazabicyclo[2.2.1] heptan-2-yl]-4-nitro- 1-(propan-2- yl)-1H-1,3- benzodiazole Method 3B And (1R,4R)-2,5- diazabicyclo[2.2.1]heptane

3.9. Compounds of Example 26

(88) ##STR00283##

5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole (Example 26A)

(89) 4,5-dibromo-1-N-(propan-2-yl)benzene-1,2-diamine (2.8 g, 9.1 mmol) and isonipeconic acid (1.17 g, 9.1 mmol) were taken up in phosphoric acid (17.82 g, 0.18 mol). The resulting mixture was stirred at 180 C. for 3.5 hours. The mixture was allowed to cool to RT and diluted with water to 200 ml. The solution was basified to pH 14.0 using solid NaOH. The resulting precipitate was then filtered off and washed repeatedly with MeOH. The filtrate was concentrated in-vacuo. The product was purified on Al.sub.2O.sub.3(basic) using DCM/MeOH/NH.sub.3 sat. in MEOH (25:15:1). The obtained product (8.7 mmol, 3.9 g) was dissolved in cone. H.sub.2SO.sub.4 (30 ml). Next KNO.sub.3 (8.7 mmol, 0.89 g) was added in one portion at 0 C. The resulting mixture was stirred at 0 C. for 3 h and at RT overnight. Then the mixture was poured onto ice. The product was filtered and washed with water. The product was purified on Al.sub.2O.sub.3 (basic) using DCM/MeOH/NH.sub.3 sat. in MEOH (25:15:1) to afford 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole (1.9 g). 1H NMR (600 MHz, DMSO) 8.74 (bs, 1H), 8.48 (s, 1H), 8.35 (bs, 1H), 4.94 (hept, J=6.8 Hz, 1H), 3.52-3.46 (m, 1H), 3.42-3.37 (m, 2H), 3.08 (bs, 2H), 2.07-1.96 (m, 4H), 1.60 (d, J=6.9 Hz, 6H). m/z 446.8; rt 2.7 min.

(90) The following compounds were prepared by the procedure of Example 26A, using the appropriate starting materials.

(91) TABLE-US-00007 1HNMR Ex. Compound (400 MHz) m/z RT SM 26B Diastereoisomer I of 3-[5,6- dibromo-4-nitro-1-(propan-2- yl)-1H-1,3-benzodiazol-2- yl]cyclohexan-1- amine 3-aminocyclohexane- 1-carboxylic acid 26C Diastereosomer II of 3-[5,6- dibromo-4-nitro-1-(propan-2- yl)-1H-1,3-benzodiazol-2- yl]cyclohexan-1- amine 3-aminocyclohexane- 1-carboxylic acid 26D 4-[5,6-dibromo-4-nitro-1- (propan-2-yl)-1H-1,3- benzodiazol-2-yl]cyclohexan- 1- amine 4-aminocyclohexane- 1-carboxylic acid

3.10. Compounds of Example 27

(92) ##STR00287##

5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(piperidin-4-ylmethyl)-1H-1,3-benzodiazole (27A)

(93) tert-butyl 4-[5,6-dibromo-1-({1-[(tert-butoxy)carbonyl]piperidin-4-yl}methyl)-1H-1,3-benzodiazol-2-yl]piperidine-1-carboxylate (Method 16A)(0.04 mmol, 20 mg) was dissolved in concentrated H.sub.2SO.sub.4 (1 ml). Then KNO.sub.3 (0.07 mmol, 6.6 mg) was added in one portion at 0 C. The resulting mixture was stirred at 0 C. for 3 h and at RT overnight. The mixture was poured onto ice. The product was purified on preparative HPLC to afford compound 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(piperidin-4-ylmethyl)-1H-1,3-benzodiazole trifluoroacetate (10 mg); m/z 502.0; rt 1.9 min.

3.11. Methods in Order to Prepare Compounds According to the Present Invention

3.11.1. Method 1

(94) ##STR00288##

2,5,6-tribromo-1H-1,3-benzodiazole

(95) 2-bromo-1H-1,3-benzodiazole (170 mmol, 33.5 g) was suspended in acetonitrile (400 ml). Then NBS (357 mmol, 63.55 g) in acetonitrile (300 ml) was added. The resulting mixture was stirred at RT until the reaction was complete (24 hours) by LC/MS. The product was filtered and washed with acetonitrile. The product was purified on silica gel using EA/hex (1:4) to afford compound 2,5,6-tribromo-1H-1,3-benzodiazole (56 g). 1H NMR (600 MHz, DMSO) 7.95 (s, 1H); m/z 356.7; rt 3.0 min.

3.11.2. Method 2A

(96) ##STR00289##

2,5,6-tribromo-4-nitro-1H-1,3-benzodiazole

(97) 2,5,6-tribromo-1H-1,3-benzodiazole (Method 1)(1.4 mmol, 500 mg) was dissolved in concentrated H.sub.2SO.sub.4 (4 ml). Then KNO.sub.3 (1.7 mmol, 171 mg) was added in one portion at 0 C. The resulting mixture was stirred at 0 C. for 3 h and at RT overnight. The mixture was poured onto ice. The product was filtered and washed with water to afford compound 2,5,6-tribromo-4-nitro-1H-1,3-benzodiazole (487 mg). 1H NMR (600 MHz, DMSO) 14.33 (s, 1H), 8.22 (s, 1H); m/z 399.7; rt 3.0 min.

(98) The following compound was prepared by the procedure of Method 2A, using the appropriate starting materials:

(99) TABLE-US-00008 1HNMR Method Compound (400 MHz) m/z RT SM 2B 2-bromo-5,6- dichloro-1-ethyl-4- nitro-1,3- benzodiazole 0 339.7 3.5 2-bromo-5,6-dichloro- 1-ethyl-1,3- benzodiazole Method 3J

3.11.3. Method 3A

(100) ##STR00292##

2,5,6-tribromo-1-ethyl-4-nitro-1H-1,3-benzodiazole

(101) 2,5,6-tribromo-4-nitro-1H-1,3-benzodiazole (Method 2) (28 mmol, 10 g) was dissolved in acetonitrile (200 ml), and then NaOH (33.8 mmol, 1.35 g) was added. The resulting mixture was stirred at temperature for 0.5 h. Next 2-iodoethane (225 mmol, 35.16 g) was added, and the mixture was heated to 85 C. until the reaction was complete (20 h) by LC/MS. The mixture was allowed to cool to RT and concentrated in-vacuo. The product was taken up into ethyl acetate and washed with water. The organic extract was dried over MgSO4, filtered and concentrated. The product was purified on silica gel using EA/hex (1:1) to afford compound 2,5,6-tribromo-1-ethyl-4-nitro-1H-1,3-benzodiazole (mg). 1H NMR (600 MHz, DMSO) 8.58 (s, 1H), 4.36 (q, J=7.2 Hz, 2H), 1.33 (t, J=7.2 Hz, 3H); m/z 427.8; rt 3.5 min.

(102) The following compounds were prepared by the procedure of Method 3A, using the appropriate starting materials:

(103) TABLE-US-00009 1HNMR Method Compound (400 MHz) m/z RT SM 3B 2,5,6-tribromo-4- nitro-1-(propan-2- yl)-1H-1,3- benzodiazole 441.7 3.7 2,5,6-tribromo-4- nitro-1H-1,3- benzodiazole: Method 2A And Isopropyl Iodide (commercial) 3C 2,5,6-tribromo-1- (cyclopropylmethyl)- 4-nitro-1H-1,3- benzodiazole 1H NMR (600 MHz, DMSO) 8.62 (s, 1H), 4.25 (d, J = 7.2 Hz, 2H), 1.35-1.27 (m, 1H), 0.55-0.51 (m, 2H), 0.50-0.48 (m, 2H). 455.8 4.0 2,5,6-tribromo-4- nitro-1H-1,3- benzodiazole: Method 2A and Methylcyclopropyl iodide (commercial) 3D 2,5,6-tribromo-1- (2-methylpropyl)- 4-nitro-1H-1,3- benzodiazole .sup.1H NMR (600 MHz, DMSO) 8.60 (s, 1H), 4.14 (d, J = 7.7 Hz, 2H), 2.24-2.14 (m, 1H), 0.90 (d, J = 6.7 Hz, 6H). 455.8 3.9 2,5,6-tribromo-4- nitro-1H-1,3- benzodiazole: Method 2A And Isobutyl Iodide (commercial) 3E 2,5,6-tribromo-1- (2-methoxyethyl)- 4-nitro-1H-1,3- benzodiazole 457.7 15.4 2,5,6-tribromo-4- nitro-1H-1,3- benzodiazole: 00 Method 2A And 1-Bromo-2- methoxyethane (commercial) 3F 2,5,6-tribromo-4- nitro-1-propyl-1H- 1,3- benzodiazole 01 1H NMR (600 MHz, DMSO) 8.60 (s, 1H), 4.28 (t, J = 7.3 Hz, 2H), 1.81-1.73 (n, 2H), 0.89 (t, J = 7.4 Hz, 3H). 441.8 3.7 2,5,6-tribromo-4- nitro-1H-1,3- benzodiazole: 02 Method 2A And 1-Propyl Iodide (commercial) 3G 2,5,6-tribromo-1- (propan-2-yl)-1H- 1,3- benzodiazole 03 1H NMR (600 MHz, DMSO) 8.25 (s, 1H), 8.04 (s, 1H), 4.91 (hept, J = 6.9 Hz, 1H), 1.58 (d, J = 7.0 Hz, 6H). 396.7 3.7 2,5,6-tribromo-1H- 1,3-benzodiazole 04 Method 1 And isopropyl iodide 3H 2,5,6-tribromo-1- ethyl-1H-1,3- benzodiazole 05 1H NMR (600 MHz, DMSO) 8.23 (s, 1H), 8.04 (s, 1H), 4.29 (q, J = 7.2 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H). 384.7 3.4 2,5,6-tribromo-1H- 1,3-benzodiazole 06 Method 1 And ethyl iodide 3J 2-bromo-5,6- dichloro-1-ethyl- 1,3- benzodiazole 07 294.7 3.4 2-bromo-5,6-dichloro- 3H-1,3- benzodiazole 08 Method 14A and ethyl iodide 3K 2,5,6-tribromo-4- nitro-1-[3-(oxan-2- yloxy)propyl]-1H- 1,3-benzodiazole 09 543.7 3.8 2,5,6-tribromo-4- nitro-1H-1,3- benzodiazole: 0 Method 2A And 2-(3- bromopropoxy)oxane (commercial) 3L 2,5,6-tribromo-4- nitro-1-[2-(oxan-2- yloxy)ethyl]- 1H-1,3- benzodiazole 527.7 3.7 2,5,6-tribromo-4- nitro-1H-1,3- benzodiazole: Method 2A And 2-(3- bromoethoxy)oxane (commercial)

3.11.4. Method 4A

(104) ##STR00313##

tert-butyl 4-(5,6-dibromo-4-nitro-1H-1,3-benzodiazol-2-yl)piperazine-1-carboxylate

(105) 2,5,6-tribromo-4-nitro-1H-1,3-benzodiazole (Method 2A)(17.5 mmol, 7 g) was dissolved in EtOH (30 ml) with N-Boc-piperazine (52.5 mmol, 9.78 g). The resulting mixture was stirred at 120 C. until the reaction was complete (8 h) by LC/MS. The mixture was allowed to cool to RT and concentrated in-vacuo. The product was purified on silica gel using DCM/MeOH (99:1) to afford tert-butyl 4-(5,6-dibromo-4-nitro-1H-1,3-benzodiazol-2-yl)piperazine-1-carboxylate (6 g). m/z 405.8; rt 2.4 min.

(106) The following compounds were prepared by the procedure of Examples 4A, using the appropriate starting materials:

(107) TABLE-US-00010 1HNMR Method Compound (400 MHz) m/z RT SM 4B tert-butyl 4-(5,6- dibromo-1-ethyl-4- nitro-1H-1,3- benzodiazol-2- yl)piperazine-1- carboxylate 1H NMR (600 MHz, DMSO) 7.88 (s, 1H), 7.78 (s, 1H), 4.09 (q, J = 7.2 Hz, 2H), 3.55-3.47 (m, 4H), 3.24-3.20 (m, 4H), 1.43 (s, 9H), 1.31 (t, J = 7.2 Hz, 3H). 533.9 3.9 2,5,6-tribromo-1- ethyl-4-nitro-1H- 1,3- benzodiazole Method 3A and tert-butyl piperazine-1- carboxylate 4C tert-butyl N-{3-[(5,6- dibromo-4-nitro-1H- 1,3-benzodiazol-2- yl)amino]propyl}carbamate 494.1 3.5 2,5,6-tribromo-4- nitro-1H-1,3- benzodiazole Method 2A and tert-butyl N-(3- aminopropyl)carbamate 4D tert-butyl N-[1-(5,6- dibromo-1-ethyl-4- nitro-1H-1,3- benzodiazol-2- yl)piperidin-3- yl]carbamate 548.0 3.9 2,5,6-tribromo-1- ethyl-4-nitro-1H- 1,3- benzodiazole Method 3A and tert-butyl N- (piperidin-3- yl)carbamate 4E tert-butyl N-{3-[(5,6- dibromo-1-ethyl-4- nitro-1H-1,3- benzodiazol-2- yl)amino]propyl}carbamate 0 522.0 3.6 2,5,6-tribromo-1- ethyl-4-nitro-1H- 1,3- benzodiazole Method 3A And tert-butyl N- (3- aminopropyl)carbamate 4F tert-butyl 4-(5,6- dibromo-1-ethyl-1H- 1,3-benzodiazol-2- yl)piperazine-1- carboxylate 489.0 3.5 2,5,6-tribromo-1- ethyl-1H-1,3- benzodiazole (Method 3H) Method 3H And tert-butyl piperazine-1-carboxylate 4G tert-butyl N-{3-[(5,6- dibromo-1-ethyl-1H- 1,3-benzodiazol-2- yl)amino]propyl}carbamate 476.9 2.8 2,5,6-tribromo-1- ethyl-1H-1,3- benzodiazole Method 3H And tert-butyl N- (3- aminopropyl)carbamate 4H tert-butyl (3S)-3-{5,6- dibromo-2-[(3R)-3- {[(tert- butoxy)carbonyl]amino} pyrrolidin-1-yl]-1H- 1,3-benzodiazol-1- yl}pyrrolidine-1- carboxylate 630.1 3.2 tert-butyl (3S)-3- (2,5,6-tribromo- 1H-1,3- benzodiazol-1- yl)pyrrolidine-1- carboxylate Method 17A

3.11.5. Method 5A

(108) ##STR00328##

1-(2,5,6-tribromo-4-nitro-1H-1,3-benzodiazol-1-yl)propan-2-ol

(109) 1,2,4-tribromo-5-nitrobenzene (10 mmol, 2.5 g) is dissolved in THF (75 ml). Triethylamine (7.6 mmol, 773 mg) and amino-2-propanol (7.6 mmol, 574 mg) were added. The resulting mixture was stirred at 45 C. until the reaction was complete (24 hours) by LC/MS. The mixture was allowed to cool to RT and concentrated in-vacuo. The product was purified on silica gel using EA/hex (1:4). The obtained product (1.5 g, 4 mmol) was suspended in a mixture of EtOH, AcOH and H.sub.2O (2:2:1), then iron filings (17 mmol, 0.947 mg) were added. The mixture was sonicated for 5 hours. The product was purified on silica gel using EA/hex (1:1). The obtained product was suspended in EtOH (30 ml), and H.sub.2O (2 ml) was added. Next potassium ethyl xanthogenate (3.8 mmol, 608 mg) was added in one portion. The resulting mixture was stirred at 85 C. until the reaction was complete (24 hours) by LC/MS. The reaction was cooled down to 60 C., and H.sub.2O (30 ml) was added, followed by addition of H.sub.2O/AcOH (2:1). The mixture was allowed to cool to RT, and solid was filtered and washed with H.sub.2O. The obtained product (2 mmol, 740 mg) was dissolved in MeOH (20 ml). The reaction mixture was cooled to 0 C. and hydrobromic acid (0.4 ml) was added, then bromine (8 mmol, 1.3 g) was added. The resulting mixture was stirred at RT overnight, then Na.sub.2SO.sub.4 was added. Next MeOH was evaporated. The aqueous layers extracted with DCM. The product was purified on silica gel using EA/hex (1:1). The obtained product (1 mmol, 400 mg) was dissolved in concentration H.sub.2SO.sub.4 (7 ml). Next KNO.sub.3 (1.1 mmol, 118 mg) was added in one portion at 0 C. The resulting mixture was stirred at 0 C. for 3 h and at RT overnight. Then the mixture was poured onto ice. The product was filtered and washed with water. The product was purified on silica gel using DCM/MeOH (95:5) to afford 2,5,6-tribromo-1-(propan-2-ol)-4-nitro-1H-1,3-benzodiazole (200 mg). m/z 457.7; rt 3.3.

(110) The following compounds were prepared by the procedure of Method 5A, using the appropriate starting materials:

(111) TABLE-US-00011 1HNMR Method Compound (400 MHz) m/z RT SM 5B 2,5-dibromo-6-fluoro- 1-(propan-2-yl)-1H- 1,3-Benzodiazole 336.8 3.4 1-bromo-2,4-difluoro- 5-nitrobenzene 0 Commercial and isopropyl amine 5C 2,5,6-tribromo-1- cyclobutyl-4-nitro-1H- 1,3-benzodiazole 453.7 3.8 1,2,4-tribromo-5- nitrobenzene Commercial and cyclobutyl amine

3.11.6. Method 7A

(112) ##STR00333##

tert-butyl 4-(5,6-dibromo-1-ethyl-4-iodo-1H-1,3-benzodiazol-2-yl)piperazine-1-carboxylate

(113) tert-butyl 4-(5,6-dibromo-1-ethyl-1H-1,3-benzodiazol-2-yl)piperazine-1-carboxylate (6 mmol, 3 g) was dissolved in dry THF (20 ml). The resulting mixture was cooled down to 78 C., then magnesium chloro-2,26,6-tetramethylpiperidine lithium chloride complex was added dropwise at this temperature. The resulting mixture was stirred at 78 C. for 2 hours. The mixture was allowed to warm to 20 C. and a1M solution of I.sub.2 in THF was added dropwise. The mixture was warmed to RT and stirred for 1.5 h. The reaction mixture was poured onto a mixture of ice/NH.sub.4Cl, then sat. Na.sub.2SO.sub.3 was added. The aqueous mixture was extracted with ethyl acetate.

(114) The organic extract was dried over MgSO4, filtered and concentrated. The product was purified on silica gel using EA/hex (1:1) to afford tert-butyl 4-(5,6-dibromo-1-ethyl-4-iodo-1H-1,3-benzodiazol-2-yl)piperazine-1-carboxylate (1.5 g). m/z 614.8; rt 4.4 min.

(115) The following compounds were prepared by the procedure of Method 7A, using the appropriate starting materials:

(116) TABLE-US-00012 1HNMR Method Compound (400 MHz) m/z RT SM 7B 2,5,6-tribromo-4-iodo- 1-(propan-2-yl)-1H- 1,3-benzodiazole 524.6 4.0 2,5,6-tribromo-1- (propan-2-yl)-1H-1,3- benzodiazole Method 3G 7C tert-butyl N-{3-[(5,6- dibromo-1-ethyl-4- iodo-1H-1,3- benzodiazol-2- yl)amino]propyl}carbamate 602.9 4.3 tert-butyl N-{3-[(5,6- dibromo-1-ethyl-1H- 1,3-benzodiazol-2- yl)amino]propyl}carbamate Method 4G 7E 2,5,6-tribromo-1- ethyl-4-iodo-1,3- benzodiazole 510.6 3.6 2,5,6-tribromo-1-ethyl- 1H-1,3-benzodiazole Method 3H 7F tert-butyl (3S)-3-{5,6- dibromo-2-[(3R)-3- {[(tert- butoxy)carbonyl]amino} pyrrolidin-1-yl]-4- iodo-1H-1,3- benzodiazol-1- yl}pyrrolidine-1- carboxylate 0 756 4.3 tert-butyl (3S)-3-{5,6- dibromo-2-[(3R)-3- {[(tert- butoxy)carbonyl]amino} pyrrolidin-1-yl]-1H- 1,3-benzodiazol-1- yl}pyrrolidine-1- carboxylate Method 4H

3.11.7. Method 8A

(117) ##STR00342##

tert-butyl 4-(5,6-dibromo-1-ethyl-4-cyano-1H-1,3-benzodiazol-2-yl)piperazine-1-carboxylate

(118) tert-butyl 4-(5,6-dibromo-1-ethyl-4-iodo-1H-1,3-benzodiazol-2-yl)piperazine-1-carboxylate (0.2 mmol, 100 mg) was dissolved in acetonitrile (1.5 ml). Then copper (I) cyanide was added. The reaction was carried out in a microwave at 160 C. for 25 min.

(119) The mixture was concentrated in-vacuo. The product was taken up into ethyl acetate and washed with water. The organic extract was dried over MgSO4, filtered and concentrated. The product was purified on silica gel using EA/hex (1:4) to afford tert-butyl 4-(5,6-dibromo-1-ethyl-4-cyano-1H-1,3-benzodiazol-2-yl)piperazine-1-carboxylate (80 mg). m/z 423.7; rt 3.3 min.

(120) The following compounds were prepared by the procedure of Method 8A, using the appropriate starting materials:

(121) TABLE-US-00013 1HNMR Method Compound (400 MHz) m/z RT SM 8B 2,5,6-tribromo-1-(propan- 2-yl)-1H-1,3- benzodiazole-4- carbonitrile 423.7 3.5 2,5,6-tribromo-4-iodo-1- (propan-2-yl)-1H-1,3- benzodiazole Method 7B 8C 2,5,6-tribromo-1-ethyl-1,3- benzodiazole-4- carbonitrile 409.7 3.2 2,5,6-tribromo-1-ethyl-4- iodo-1,3-benzodiazole Method 7E 8D tert-butyl (3S)-3-{5,6- dibromo-2-[(3R)-3-{[(tert- butoxy)carbonyl]amino} pyrrolidin-1-yl]-4-cyano-1H- 1,3-benzodiazol-1- yl}pyrrolidine-1- carboxylate 654.9 4.3 tert-butyl (3S)-3-{5,6- dibromo-2-[(3R)-3-{[(tert- butoxy)carbonyl]amino}pyrrolidin- 1-yl]-4-iodo-1H-1,3- benzodiazol-1- yl}pyrrolidine-1- carboxylate Method 7F

3.11.8. Method 13

(122) ##STR00349##

2,5-dibromo-6-fluoro-4-nitro-1-(propan-2-yl)-1H-1,3-benzodiazole

(123) 2,5-dibromo-6-fluoro-1-(propan-2-yl)-13.11H-1,3-benzodiazole (Method 5B) (0.15 mmol, 50 mg) was dissolved in TFA (0.5 ml). Then HNO.sub.3 (2.9 mmol, 0.12 ml) was added slowly at RT. The resulting mixture was stirred at RT for overnight. The mixture was poured onto ice. The product was filtered and washed with water to afford compound 2,5,6-tribromo-4-nitro-1H-1,3-benzodiazole (487 mg). 1H NMR (600 MHz, DMSO) 14.33 (s, 1H), 8.22 (s, 1H); m/z 381; rt 3.4.

3.11.9. Method 14A

(124) ##STR00350##

2-bromo-5,6-dichloro-1H-1,3-benzodiazole

(125) 5,6-dichloro-2,3-dihydro-1H-1,3-benzodiazole-2-thione (2 mmol, 438 mg) was dissolved in MeOH (20 ml). The reaction mixture was cooled to 0 C. and hydrobromic acid (0.4 ml) was added, then bromine (8 mmol, 1.3 g) was added. The resulting mixture was stirred at RT overnight, then Na.sub.2SO.sub.4 was added. Next MeOH was evaporated. The aqueous layer was extracted with DCM. The product was purified on silica gel using EA/hex (1:1) to afford yellow solid (1 mmol, 260 mg), m/z 266.7; rt 2.8 min.

3.11.10. Method 15A

(126) ##STR00351##

(127) 1,2,4-tribromo-5-nitrobenzene (14 mmol, 5 g) was dissolved in i-PrOH (130 ml). Triethylamine (15.3 mmol, 1.55 g) and 2-aminopropane (15.3 mmol, 0.91 g) were added. The resulting mixture was stirred at 90 C. until the reaction was complete (24 hours) by LC/MS. The mixture was allowed to cool to RT and concentrated in-vacuo. The product was purified on silica gel using EA/hex (1:4). The obtained product (3.33 g, 9.85 mol) was suspended in a mixture of EtOH, AcOH and H.sub.2O (2:2:1), then iron filings (49.3 mmol, 2.75 g) were added. The mixture was sonicated for 5 hours. The product was purified on silica gel using EA/hex (1:1). yield (2.8 g) m/z 308, rt. 3.2 min.

3.11.11. Method 16A

(128) ##STR00352##

tert-butyl 4-[5,6-dibromo-1-({1-[(tert-butoxy)carbonyl]piperidin-4-yl}methyl)-1H-1,3-benzodiazol-2-yl]piperidine-1-carboxylate (16A)

(129) 4,5-dibromobenzene-1,2-diamine (100 mg, 0.38 mmol) and tert-butyl 4-formylpiperidine-1-carboxylate (160 mg, 0.76 mmol) were stirred for 1 hour in 2,2,2trifluoroethanol. Then Solvent was evaporated and product was purified on silica gel using EA/hex (1/1). Yield: 20 mg. m/z 657.1, rt. 4.2 min.

3.11.12. Method 17A

(130) ##STR00353##

tert-butyl (3S)-3-(2,5,6-tribromo-1H-1,3-benzodiazol-1-yl)pyrrolidine-1-carboxylate (17A)

(131) 1,2,4-tribromo-5-nitrobenzene (4.4 mmol, 1.6 g) is dissolved in THF (75 ml). Triethylamine (7.6 mmol, 773 mg) and tert-butyl (3S)-3-aminopyrrolidine-1-carboxylate (4.4 mmol, 828 mg) were added. The resulting mixture was stirred at 100 C. until the reaction was complete (72 hours) by LC/MS. The mixture was allowed to cool to RT and concentrated in-vacuo. The product was purified on silica gel using EA/hex (1:4). The obtained product (1.86 g, 4 mmol) was suspended in a mixture of EtOH, AcOH and H.sub.2O (2:2:1), then iron filings (17 mmol, 0.947 mg) were added. The mixture was sonicated for 5 hours. The product was purified on silica gel using EA/hex (1:1). The obtained product was suspended in EtOH (30 ml), and H.sub.2O (2 ml) was added. Next potassium ethyl xanthogenate (3.8 mmol, 608 mg) was added in one portion. The resulting mixture was stirred at 85 C. until the reaction was complete (24 hours) by LC/MS. The reaction was cooled down to 60 C., and H.sub.2O (30 ml) was added, followed by addition of H.sub.2O/AcOH (2:1). The mixture was allowed to cool to RT, and solid was filtered and washed with H.sub.2O. The obtained product (2 mmol, 954 mg) was dissolved in MeOH (20 ml). The reaction mixture was cooled to 0 C. and hydrobromic acid (0.4 ml) was added, then bromine (8 mmol, 1.3 g) was added. The resulting mixture was stirred at RT overnight, then Na.sub.2SO.sub.4 was added. Next MeOH was evaporated. The aqueous layers extracted with DCM. The product was purified on silica gel using EA/hex (1:1). Yield 200 mg, m/z: 523.8, rt: 3.2 min.

3.12. Determination of the Inhibitory Activity In Vitro

(132) Compounds of the present invention were tested for their inhibitory activity against Pim-1, Pim-2, Pim-3, Flt3 wt, Flt3 ITD, CDK2/E and DYRK1. The testing of the compounds was carried out using the ADP-Glo Kinase Assay from Promega Corporation (Madison, Wis., USA). Percent inhibition at 1 M concentration was determined for the compounds and the results are shown in Table 1A.

(133) The ADP-Glo Kinase Assay is a luminescent ADP detection assay to measure kinase activity by quantifying the amount of ADP produced during a kinase reaction. The kinase assay is performed in kinase assay buffer (5 mM MOPS, pH 7.5, 5 mM MgCl.sub.2, 0.4 mM EDTA, 1.5 mM DTT). Test samples initially dissolved in DMSO at 10 mM were diluted with the assay buffer to 1000 nM. A 30 L volume/well of a mixture of substrates containing ATP (final ATP concentration in each kinase assay was equal to its apparent ATP Km).

(134) Pim-1 (Biocentrum, Krakw, Poland) was used at the concentration of 3 ng/well and the peptide KKRNRTLTV (Lipopharm, Gdask, Poland) was used as a substrate at the concentration of 80 M, the determined Km ATP was 50 M.

(135) Pim-2 (Biocentrum, Krakw, Poland) was used at the concentration of 120 ng/well and the peptide RSRHSSYPAGT (Lipopharm, Gdask, Poland) was used as a substrate at the concentration of 10 M, the determined Km ATP was 6 M.

(136) Pim-3 (Biocentrum, Krakw, Poland) was used at the concentration of 80 ng/well and the peptide KKRNRTLTV (Lipopharm, Gdask, Poland) was used as a substrate at the concentration of 150 M, the determined Km ATP was 36.6 M.

(137) Flt3 wt (Carna Bioscience, Kobe, Japan) was used at the concentration of 75 ng/well and the peptide EAIYAAPFAKKK (Lipopharm, Gdask, Poland) was used as a substrate at the concentration of 40 M, the determined Km ATP was 65 M.

(138) FLT3-ITD (Human FLT3, C-terminal fragment, amino acids R571-S993; Product No.: 0778-0000-1, Proqinase, Germany) was used at the concentration of 70 ng/well, the peptide EAIYAAPFAKKK (Lipopharm, Gdask, Poland) was used as a substrate at the concentration of 250 M, the determined Km ATP was 70 M.

(139) CDK2/E (Millipore Billerica, Mass., USA) was used at the concentration of 20 ng/well and the peptide PKTPKKAKKL (Lipopharm, Gdask, Poland) was used as a substrate at the concentration of 108 M, the determined Km ATP was 130 M.

(140) DYRK1 (Milipore, Billerica, Mass., USA) was used at the concentration of 50 ng/well and the peptide KKISGRLSPIMTEQ (Lipopharm, Gdask, Poland) was used as a substrate at the concentration of 36 M, the determined Km ATP was 35 M.

(141) The assay was performed in two steps: first, after the kinase reaction, an equal volume of ADP-Glo Reagent was added to terminate the kinase reaction and deplete the remaining ATP. Second, the Kinase Detection Reagent was added to simultaneously convert ADP to ATP and allowed the newly synthesized ATP to be measured using a luciferase/luciferin reaction. The luminescent signal generated was proportional to the ADP concentration produced and was correlated with kinase activity. A microplate spectrophotometer (Synergy 2 multi-mode microplate reader [BioTek]) was used for detecting the luminescence. The data was normalized and the percent of inhibition was obtained according to the following equation:

(142) $\%\mathrm{inhibition}=100\%-\left(\frac{{\mathrm{Lum}}_{\mathrm{Cpd}}100\%}{{\mathrm{Lum}}_{\mathrm{PC}}}\right)$$\begin{array}{c}\%\mathrm{inhibition}\text{-}\mathrm{percent}\mathrm{of}\mathrm{inhibition}\\ {\mathrm{Lum}}_{\mathrm{Cpd}}\text{-}\mathrm{value}\mathrm{of}\mathrm{compound}s\mathrm{luminescence}\left(\mathrm{in}\mathrm{RLU}\right)\\ {\mathrm{Lum}}_{\mathrm{PC}}\text{-}\mathrm{value}\mathrm{of}\mathrm{positive}\mathrm{control}s\mathrm{luminescence}\left(\mathrm{in}\mathrm{RLU}\right)\end{array}$

(143) TABLE-US-00014 TABLE 1A In vitro inhibitory activity of compounds of the present invention Pim-1 Pim-2 Pim-3 Flt3wt Flt3ITD CDK2/E DYRK1A % INH % INH % INH % INH % INH % INH % INH Ex. (1 M) (1 M) (1 M) (1 M) (1 M) (1 M) (1 M) 1A 81 22 65 26 81 <5 66 1AA >95 53 >95 11 <5 40 1AB >95 46 87 <5 <5 1AC 94 18 72 <5 <5 1AD >95 52 >95 18 26 72 1AE 91 20 >95 <5 <5 66 1AF >95 23 71 <5 <5 47 1AG >95 51 77 11 43 62 1AH >95 55 >95 9 29 6 39 1AI >95 22 >95 <5 9 13 1AJ >95 41 83 <5 28 37 1AK 92 17 >95 <5 16 75 1AL >95 42 >95 <5 <5 49 1AM 85 53 <5 12 1AN 80 39 88 <5 <5 19 1AO 95 59 26 15 1AP >95 80 93 56 13 70 1AQ >95 40 83 <5 <5 22 1AR >95 30 52 <5 <5 13 1AS >95 77 93 22 <5 70 1AT >95 85 >95 45 <5 45 1AU >95 52 68 7 <5 1AV >95 54 91 37 12 46 1AW >95 38 73 <5 <5 37 1AX >95 77 95 83 96 >95 1AY >95 89 >95 60 87 1AZ >95 70 94 48 38 72 1BA >95 51 82 15 6 52 1BB >95 12 65 33 29 61 1BC 79 16 46 <5 <5 14 1BD >95 65 88 28 <5 53 1BG >95 82 >95 23 <5 >95 1BH 83 13 60 <5 <5 22 1BI >95 82 95 27 22 79 1BJ >95 82 >95 31 <5 54 1BK >95 63 92 20 <5 48 1BL >95 33 81 54 7 75 1BM >95 62 <5 <5 1BN >95 29 77 <5 <5 21 1BS >95 21 48 39 21 66 1BT 70 16 56 23 26 22 1BV >95 82 91 39 11 80 1C 85 28 70 <5 <5 1CA 77 64 1CD >95 62 72 5 <5 19 1CE >95 53 76 12 <5 37 1CH 82 10 32 17 9 14 1CI >95 64 75 19 10 57 1CK >95 30 24 23 1CL 82 45 1CM >95 26 95 1CN 76 27 1CO >95 25 69 12 27 30 1CP >95 32 39 44 1CQ >95 17 50 28 1CR >95 44 70 1CS >95 23 56 10 9 37 1CT >95 28 64 7 6 22 1CX >95 44 73 1CY >95 <5 1D >95 41 57 <5 21 16 1DA 91 36 1DC >95 52 >95 1DF >95 18 55 1DG >95 92 1DJ 95 >95 1DK >95 25 1DN >95 55 38 1DO 93 1DP >95 57 >95 1DQ >95 53 86 <5 <5 38 1DR >95 59 91 18 50 28 65 1DS >95 54 8 47 23 1DT >95 <5 46 34 1DU >95 12 53 39 1DV >95 <5 50 95 1DW >95 8 48 1DY >95 18 62 <5 7 20 1DZ >95 1EA >95 1EB >95 1F 90 36 83 6 <5 20 1G 76 34 57 6 <5 12 1H 58 25 38 <5 <5 9 1L >95 58 >95 21 32 47 1M >95 44 73 46 <5 >95 1N >95 69 >95 55 39 >95 1P >95 36 77 37 <5 23 1Q 86 22 >95 <5 6 11 1R >95 61 >95 54 25 >95 1T >95 81 >95 21 7 27 1U >95 37 >95 9 <5 35 1V >95 >95 <5 <5 11 1W 89 92 <5 <5 40 1X >95 36 >95 <5 17 16 1Y >95 25 86 <5 6 1Z 67 92 <5 <5 21A 87 42 71 37 5 50 22A 65 28 8 <5 22AA >95 36 88 49 11 43 22AB >95 31 76 <5 <5 17 22AC >95 30 61 7 <5 16 22AD >95 31 22AE 76 95 22G >95 47 87 13 5 22H >95 71 <5 16 22I >95 28 84 <5 <5 20 22J >95 31 79 <5 5 <5 16 22K >95 40 85 23 <5 28 22L 80 >95 <5 <5 17 22M 74 18 29 <5 <5 22N 82 23 44 <5 <5 21 22W 80 57 <5 <5 22X 74 56 <5 <5 39 22Z >95 37 85 73 63 26A >95 68 90 65 93 36 >95 26B >95 43 36 26C >95 55 55 26D >95 38 36 27A 56 2A >95 49 89 66 93 44 >95 2B 53 <5 <5 61 2D >95 53 >95 55 21 >95 2E 79 63 56 25 15 72 2F >95 70 89 78 15 >95 2G 71 34 10 <5 25 2H >95 56 >95 <5 <5 62 2I >95 75 >95 89 60 >95 2L 67 3A >95 <5 20 3B >95 18 4A 81 4B >95 51 8A 82 14 92 11 <5 46 8G >95 9A >95 15 66 76 <5 77 9B 59 33

(144) Based on the activity shown in the in vitro tests, the compounds of the present invention are useful PIM-kinase inhibitors since they inhibit Pim-1 to a high degree (>50% when tested at 1 M). The compounds according to the present invention also inhibit Pim-2 and Pim-3 to a rather high degree. Some of the compounds inhibit Flt3 wt, whereas others do not show an inhibitory activity against Flt3 wt. The compounds of the present invention fail to substantially inhibit CDK2/E, whereas the compounds of the present invention display a rather strong inhibitory efficacy against DYRK1.

(145) Selected compounds were also tested for their binding properties against FLT3 kinase mutants using suitable in vitro assays (performed according to standard assays at DiscoveRx Corporation). The compounds show strong binding to the main oncogenic mutants of the FLT3 kinase, see Table 1B.

(146) TABLE-US-00015 TABLE 1B Binding activity of compounds 1A and 2A towards FLT wildtype and kinase mutants Kd[nM] 1A 2A FLT3wt 400 130 FLT3(ITD) 74 18 FLT3(D835H) 120 28 FLT3(D835Y) 46 15

3.13. Determination of the Growth Inhibitory Activity in Cancer Cell Lines

(147) The following cell lines, were obtained and used in tests as outlined below: Human myelomonocytic, biphenotypic leukemia MV4-11 cells (harboring a Flt3-ITD mutation); Human Acute Myeloid Leukemia MOLM16 cells; Human Acute Myeloid Leukemia MOLM13 cells (harboring a Flt3-ITD mutation); Human Myeloid Leukemia KG-1 cells; Human erythroleukemia HEL92 cells; Human mantle cell lymphoma Jeko-1 cells; Human hepatocellular carcinoma HepG2 cells; and Human colon adenocarcinoma SW-480 cells.

(148) The assays were carried out according to the following protocol, which is described as an example for the MV4-11 cells:

(149) Ten thousand MV4-11 cells were inoculated into each well of a 96-well microplate (manufactured by Corning Corp.) using Iscove's MDM medium (culture medium) containing 10% fetal calf serum (FCS). The same day, a dimethyl sulfoxide (DMSO) solution of each test compound prepared in a concentration of 10 mmol/L was further diluted with DMSO to the desired concentrations (0.1, 0.5, 1, 2.5, 5 and 10 micromol/L), and the diluted solution was added to each well. The individual wells were further cultured in 5% carbon dioxide at 37 C. for 72 hours. Following this incubation, a standart MTS assay according to the Manufacturer's instructions (CellTiter96 AQueous One Solution Cell Proliferation Assay, Promega) was performed. Briefly, 10 l MTS (3-(4,5-Dimethyl-2-thiazolyl)-5-(carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium was added to each well, and culturing was performed in 5% carbon dioxide at 37 C. for 2 hours. After this incubation and using a microplate spectrophotometer (Synergy 2 multi-mode microplate reader (BioTek)), the absorbance of each well was measured at 490 nm. The value for cells not incubated with a test compound was designated as 100%. By comparing these values with the absorbance difference obtained at the well in which each test compound was added, the cell viability (% viability) after treatment with the test compound was calculated. The results are shown in Table 2.

(150) TABLE-US-00016 TABLE 2 Inhibitory activity of compounds of the present invention on oncogenic cell growth MV4-11 HEL92 HepG2 Jeko-1 SW-480 MOLM 16 MOLM 13 KG-1 ED50 ED50 ED50 ED50 ED50 ED50 ED50 ED50 Ex. (M) (M) (M) (M) (M) (M) (M) (M) 1A 0.5 2.6 1.3 0.8 2.5 0.5 0.7 1AA 1.3 4.6 2.1 1.8 5.5 1AB 2.8 5.8 2.3 1.2 3.2 1AC 1.5 5.0 1.5 0.6 2.7 1AD 0.5 0.5 0.6 0.4 0.7 1AE 0.7 5.8 1.6 2.6 3.8 1AF 1.2 8.5 1.8 2.3 5.2 1AG 2.0 3.2 2.0 1.4 2.7 1AH 0.6 6.0 2.2 1.9 7.0 0.2 1.8 0.65 1AI 1.1 3.8 1.4 3.1 4.6 1AJ 0.9 3.1 1.8 1.6 2.9 1AK 7.5 1AL 2.2 5.3 1.4 2.3 5.3 1AM 2.6 8.0 2.7 2.8 1AO 0.6 3.8 1.7 1AP 0.3 2.4 0.9 1.3 2.4 1AQ 2.5 9.6 4.9 1AR 7.8 1AS 0.6 5.4 1.4 1.1 1AT 1.0 5.2 2.0 1AU 2.4 5.8 1.9 1AV 1.4 4.5 1AW 4.5 4.2 1AX 0.1 0.1 0.1 >0.1 0.02 0.06 0.12 1AY 0.5 1.9 1.5 0.9 1AZ 1.1 2.6 2.2 1BA 1.1 6.9 1.9 1BB 0.7 5.4 2.0 3.0 1BC 3.2 7.9 3.0 1BD 0.7 5.5 3.3 1.6 2.1 1BF 2.0 7.6 2.3 1BG 1.6 4.0 1BH 4.6 5.6 1BI 0.6 5.1 1.5 0.7 0.08 3.25 0.09 1BJ 0.6 4.8 1BK 0.8 5.2 0.06 1BL 0.4 2.4 1.4 1.0 1BM 4.9 4.5 1BN 1.9 5.1 1BS 0.9 4.6 3.6 1BT 0.5 5.7 1.2 1BV 5.1 5.3 2.9 1C 3.1 >10 4.1 7.4 1CA 2.6 9.7 1.8 1CD 2.4 5.5 5.3 1CE 2.8 3.2 1CH 5.0 5.7 2.2 1CI 1.6 6.1 1.4 1CJ 5.5 5.5 5.3 1CK 2.0 6.9 5.3 1CL 1.0 2.4 1.5 1CM 0.8 5.2 2.6 1CN 3.3 9.4 4.5 1CO 4.3 3.9 1CP 0.6 5.8 1.2 1CQ 1.7 6.0 2.0 1CR 1.1 8.7 1.9 1CS 4.8 4.7 1CT 2.7 6.0 1CX 1.2 4.1 3.1 1CY 2.1 8.9 2.8 1D 5.9 8.2 6.2 6.2 1DA 0.5 1.1 0.9 1DC 0.2 2.0 1.0 0.16 1DF 2.0 3.7 1DG 0.2 1.8 0.4 1DJ 0.6 6.7 2.1 1DK 5.5 5.8 5.7 1DN 0.5 4.5 0.9 1DO 4.3 2.2 1DP 0.1 0.5 0.2 1DQ 0.9 7.6 1.4 1.6 0.1 2.8 0.5 1DR 1.0 6.2 1.2 1.3 0.1 3.9 0.14 1DS 0.8 5.8 0.6 1.4 0.32 9.9 0.2 1DT 1.5 1.0 0.3 1DU 1.0 5.5 1.3 0.9 1DV 2.1 2.9 1.5 1DW 5.1 0.3 1DY 2.3 2.8 1.1 6.4 1.9 1E 3.3 6.8 8.1 1F 2.3 5.0 3.3 3.3 1H 2.3 7.6 2.7 3.4 1I 5.1 4.3 5.6 1L 1.2 5.7 1.6 3.3 1M 0.6 2.2 1.8 2.1 1N 0.6 1.4 1.7 1.2 1P 1.4 7.0 1.5 2.9 1Q 6.9 8.2 1.1 5.4 5.3 1R 0.6 1.5 1.1 0.5 1.4 1S 3.0 >10 3.3 8.9 8.5 1T 0.6 5.1 1.3 1.7 3.2 1U 0.6 4.7 1.5 1.8 3.3 1V 0.7 5.6 0.9 2.9 3.1 1X 1.2 4.3 1Y 1.4 3.4 1Z 1.7 6.4 5.6 6.9 21A 0.5 1.9 1.6 0.6 3.2 22AA 0.5 1.2 1.8 22AB 3.2 5.1 22AC 4.1 7.2 22AD 0.7 6.2 1.2 22AE 2.7 >10 2.8 22B 7.9 8.8 >10 3.0 6.0 22G 4.8 22I 5.4 22J 2.8 4.7 22K 4.1 22L 1.9 3.2 1.3 1.2 22M 3.7 6.5 5.0 4.7 22N 2.6 4.2 22O 2.8 2.5 1.4 1.4 22P 2.9 5.9 2.7 2.5 22R 1.1 2.9 1.2 0.5 22W 1.7 9.5 1.5 4.9 22X 0.7 8.7 2.3 5.0 22Z 0.7 2.4 1.6 26A 0.2 0.9 0.6 0.3 0.22 0.32 0.47 26B 0.4 6.1 0.1 26C 0.1 3.0 0.2 26D 0.8 9.7 1.0 2A 0.3 0.7 0.6 0.4 0.8 0.14 0.25 0.63 2B 3.1 6.7 1.4 5.5 2C 2.8 7.0 6.2 2D 0.6 4.7 0.7 0.6 2E 5.5 5.4 0.9 1.6 1.8 2F 0.3 2.1 1.8 2G 2.3 6.0 1.3 3.1 2H 2.0 6.8 1.5 1.4 2I 0.2 0.8 1.3 2L 6.1 3.1 3A 2.3 6.3 1.9 3B 1.3 1.5 4A 1.0 8.7 4B 1.2 4.1 1.8 8A 1.3 4.8 1.7 1.4 4.7 9A 0.3 1.5 9B 5.9 2.7 7.29

(151) If a compound exhibits an ED50<10 M, the compound is regarded as efficiently inhibiting the cell growth. The assays establish that the compounds according to the present invention are effective in inhibiting oncogenic cell growth in human cancer cell lines as described above.

3.14. Analysis of Pim-Kinase Biomarkers in Response to Cell Treatment with Compounds of the Present Invention

(152) The efficacy of compounds 1A and 2A on Pim kinase-inhibition was tested in MV4-11 cells (see above). Cells were treated with each compound at concentrations of 0.25, 0.5, 1, 2.5 and 5 M for 4 and 24 h. The positive control Ref A (the commercially available inhibitor SGI-1776 [obtained from Selleck Bio]) was used at a concentration of 5 M. DMSO (Dimethyl sulfoxide) was used as a negative control. The levels of the following classical Pim-1 kinase biomarkers were assessed: c-Myc, phospho-4EBP1 (Ser65, Thr37&46) and phosphorylated S6 (Ser235). The levels of c-myc protein, phosphorylated 4EBP1 and pS6 were downregulated both after 4 and 24 hours of treatment, in a dose-dependent manner (in the test using compound 2A, pS6 phosphorylation is increasing again at higher concentrations and longer incubation times; this effect is unspecific and due to massive apoptosis, which can be recognized from drastically increased PARP-cleavage). Also, the levels of pro-apoptotic and pro-survival biomarkers were assessed. First, induction of apoptosis, recognizable as an appearance and increased expression of cleaved form of the PARP protein, was observed, both at 4 and 24 hours after compound stimulation at high concentrations. Analysis of Mcl-1, a pro-survival protein, showed dosed-dependent protein down-regulation after 4 and 24 hours. Levels of tubulin were assessed as a reference loading control. Further, the levels of phosphorylated p44/42 (Erk1/2) as Flt3 biomarker were assessed; as can be derived from FIGS. 1 and 2, the levels of phosphoryled p44/42 were also downregulated.

(153) The results for compound 1A are shown in FIG. 1, whereas the results for compound 2A are shown in FIG. 2.

(154) The efficacy of compound 1BI on Pim kinase-inhibition was tested in MV4-11 cells. The cells were treated with compound 1BI at concentrations of 0.25, 0.5, 1, 2.5 and 5 M for 4 and 24 h. The positive controls Ref. A (SGI-1776, see above) and Ref. B (the commercially available inhibitor Sunitinib [obtained from Ark Pharm]) were used at 5 M concentration. DMSO (Dimethyl sulfoxide) was used as a negative control. The levels of the following classical Pim-1 kinase biomarkers were assessed: c-myc, phospho-4EBP1 (Ser65, Thr37&46) and phosphorylated S6 (Ser235/236). The levels of c-myc protein and phosphorylated 4EBP1 (Ser65, Thr37/46 at higher concentrations and 24 h) were down-regulated both after 4 and 24 hours of treatment, in a dose-dependent manner. The levels of phosphorylated S6 were diminished almost completely both after 4 and 24 hours of treatment in all concentrations. The levels of pro-apoptotic and pro-survival biomarkers were also assessed. First, induction of apoptosis, presented as an appearance and increased expression of cleaved form of PARP protein, was observed in the highest concentration at 4 hours after compound stimulation. Analysis of Mcl-1, a pro-survival protein, showed dosed-dependent protein down-regulation after 4 and 24 hours. Levels of tubulin were assessed as a reference loading control. Further, the levels of phosphorylated p44/42 (Erk1/2) as Flt3 biomarker were assessed; as can be derived from FIG. 3, the levels of phosphoryled p44/42 were also downregulated.

(155) The results for compound 1BI are shown in FIG. 3.

(156) The efficacy of compound 1BI on Pim kinase-inhibition was also tested in MOLM16 cells (an acute myeloid leukemia cell line). The cells were treated with compound 1BI at concentrations of 0.1, 0.25, 0.5, 1 and 2.5 M for 4 and 24 h. The positive controls Ref. A (SGI-1776, see above) and Ref. B (Sunitinib, see above) were used at 5 M concentration. DMSO (Dimethyl sulfoxide) was used as a negative control. The levels of the following classical Pim-1 kinase biomarkers were assessed: c-myc, phospho-4EBP1 (Scr65, Thr37&46) and phosphorylated S6 (Scr235/236). The levels of c-myc protein and phosphorylated 4EBP1 (Ser65 and Thr37/46) were downregulated both after 4 and 24 hours of treatment, in a dose-dependent manner. The levels of phosphorylated S6 were diminished completely both after 4 and 24 hours of treatment in all concentrations. The levels of a pro-apoptotic biomarker were assessed; induction of apoptosis, presented as an appearance and increased expression of cleaved form of PARP protein, was observed. Levels of tubulin were assessed as a reference loading control.

(157) The results for compound 1A in MOLM-16 cells are shown in FIG. 4.

(158) The above analysis clearly establishes that the compounds according to the present invention are capable of inhibiting PIM-kinases in vivo since downstream PIM-kinase targets are clearly affected.

3.15. Determination of In Vivo Activity Against Xenograft Tumors Implanted in Immunosuppressed Animals

(159) Several compounds of the present invention have been studied in a xenograft in mice, an in vivo tumor transplantation model used to investigate the factors involved in malignant transformation, invasion and metastasis, as well as to examine response to therapy. For the purpose of acceptance of donor leukemic cells (MV4-11 or MOLM16 cells), immunocompromised mice were used, namely particularly severely compromised immunodeficient mice (NOD/scid, SCID/beige). When tumors developed size of approx. 50-200 mm.sup.3, the compounds as indicated below in Tables 3 and 3a were administered orally every day for 2-3 weeks, in once a day (QD) or twice a day (BID) schedule. During the course of the experiment, the mice were monitored and the following two parameters were measured: the tumor growth inhibition (TGI) factor as a measure of therapeutic efficacy and the body weight change (BW) factor as a measure of possible compound toxicity. The results are depicted in Tables 3 and 3a.

(160) TABLE-US-00017 TABLE 3 MV4-11 xenograft results. TGI BW mg/Kg Example [%] [%] admin. Dosing Comment 1A 73 5.4 75 BID 1N 52 6.5 150 QD 2A 99 4.4 150 QD discontinued after 8 days due to remissions 1M 76 9.2 150 BID 2D 88 3.5 150 QD 1R 61 8 150 QD 1AH 74 0.6 150 QD 1AP 99 11.7 150 QD 1AX 97 2.2 100 QD 1AZ 76 0.2 150 QD 1BI 87 3.8 150 QD TGItumor growth inhibition, BWbody weight change, QDonce a day, BIDtwice a day.

(161) Among the tested compounds, compounds 2A, 1AP and 1AX showed the best anti-cancer activity with TGI exceeding 97%. Compounds 2D and 1BI showed very good TGI above 87%. Compounds 1A, 1M, 1AH and 1AZ led to more than 70% inhibition of tumor growth and may thus be classified as compounds with good efficacy. Compounds 1N and 1R showed moderate TGI reaching up to 70%. All tested compounds did not cause major toxicity as assessed by monitoring of the body weight change. If body weight loss was observed, this loss did not exceed 10% such that all compounds were regarded as being not toxic.

(162) Additionally, compound 2A, together with other examples was tested in MOLM16 cells xenografted into immunocompromised mice. One of the obtained results is presented below. The treatment with compound 2A resulted in >99% inhibition of the tumour growth as can be derived from Table 3a and FIG. 5.

(163) TABLE-US-00018 TABLE 3a MOLM16 xenograft results. TGI BW mg/Kg Example (%) (%) admin. Dosing Comment 2A >99 8.6 100 QD TGItumor growth inhibition, BWbody weight change, QDonce a day.

(164) Next, compound 26A was evaluated in a xenograft study of acute myeloid leukemia (MV-4-11), alone or in a combinational treatment with Cytarabine in vivo (Table 3b; FIG. 6). Compound 26A was tested in two doses (50 and 25 mg/kg) and administered twice a day (BID); Cytarabine was administered at dose of 50 mg/kg three times in a week (TIW). During 15 days of compound administration dose-dependent anti-cancer activity of compound 26A (administered alone) was shown. Tumor growth inhibition reached 82% and 77%, respectively. In addition, combinational treatment with Cytarabine showed synergistic effects, similarly dependend on the dose, and resulted in 99% and 89% TGI. Treatment with Cytarabine alone resulted in moderate, 60% inhibition of tumor growth.

(165) TABLE-US-00019 TABLE 3b MV-4-11 xenograft results. TGI BW mg/Kg Compounds (%) (%) admin. Dosing Comment 26A 87 3 50 BID 26A 77 6 25 BID Cytarabine 60 2 50 TIW 26A + 99 4 50/50 BID/TIW Cytarabine 26A + 89 2 50/25 BID/TIW Cytarabine TGItumor growth inhibition, BWbody weight change, BIDtwice a day, TIWthree times a week.

3.16. Synergistic and Additive Interactions with Anti-Cancer Agents

(166) In order to determine the efficacy of the compounds of the present invention on cancer cell growth inhibition in combination with commercially available anti-cancer agents, compounds 1A and 26A were added in combination with an anti-cancer agent to cells as indicated in Table 4. The anti-cancer agents are also indicated in Table 4.

(167) The combinations were studied at fixed concentrations, wherein compound 1A or compound 26A were tested at two constant concentrationsone corresponding to ED50 value (for compound 1A in the specified cell line (i.e. for HEL-92: 5.46 M; U-937: 6.64 M; MV4-11: 0.50 M; PC3: 2.91 M, Mino: 1.7 M); for compound 26A in MV4-11: 0.1 M; MOLM-16: 0.4 M and one below the ED50 value e.g. twice as low (see Table 4), while the therapeutic agents indicated in Table 4 were tested in a range of six increasing concentrations (Table 4). The cells were incubated with the combination of compounds for 72 hours. After this incubation, a cell viability assay was carried out according to the Manufacturer's instructions (CellTiter 96AQueous Non-Radioactive Cell Proliferation Assay, Promega). The results were expressed as percentage of viable cells upon treatment with the individual drugs or the combination compared to the vehicle (DMSO) treated cells.

(168) Based on these data, combination index (CI) values were determined using CompuSyn Software (ComboSyn Software Incorporated, Paramus, N.J.). In order to indicate the effect of combinations, the following guidelines were implemented: CI value<1 indicates synergism, CI value=1 indicates additive effect and CI value>1 indicates antagonism.

(169) TABLE-US-00020 TABLE 4 Combinations study - Examples 1A and 26A. Concentrations of drug Compound Drug [M] Cell line Effect 1A Rapamycin 0.0005; 0.001; 0.0025; PC3 Synergistic 0.005; 0.01; 0.025 (Prostate cancer) 1A Wortmannin 0.1; 0.25; 0.5; 1.0; 2.5; PC3 Synergistic 5.0 (Prostate cancer) 1A GDC-0941 0.01; 0.025; 0.05; 0.1; PC3 Synergistic/ 0.25; 0.5 (Prostate cancer) Additive 1A CP690550 0.25; 0.5; 1.0; 2.5; 5.0; HEL92 Synergistic 10 (Erythroleukemia) 1A Cyt387 0.25; 0.5; 1.0; 2.5; 5.0; HEL92 Synergistic 10 (Erythroleukemia) 1A Ruxolitinib 0.25; 0.5; 1.0; 2.5; 5.0; HEL92 Synergistic/ 10 (Erythroleukemia) Additive 1A Obatoclax 0.05; 0.1; 0.5; 1.0; 2.5; U937 Synergistic 5.0 (Histiocytic lymphoma) 1A ABT737 0.1; 0.5; 1.0; 2.5; 5.0; U937 Synergistic 10 (Histiocytic lymphoma) 1A CAL-101 0.25; 0.5; 1.0; 2.5; 5.0; MV4-11 Synergistic 10 (Acute myeloid leukemia) 1A CAL-101 0.25; 0.5; 1; 2.5; 5; 10 PC3 Synergistic (Prostate cancer) 1A PD0332991 0.005; 0.01; 0.025; Mino (Mantle cell Synergistic 0.05; 0.1; 0.25 lymphoma) 26A C 0.1; 0.25; 0.5; 1.0; 2.5; MV4-11 (Acute Synergistic 5.0 myeloid leukemia) 26A C 0.01; 0.1; 1.0; 2.5; 5.0; MOLM-16 (Acute Synergistic 10.0 myeloid leukemia) 26A V 0.01; 0.027; 0.067; MV4-11 (Acute Synergistic 0.0168, 0.42; 1.05 myeloid leukemia) 26A V 0.022; 0.054; 0.136; MOLM-16 (Acute Synergistic 0.34; 0.85; 2.13 myeloid leukemia)

(170) The above results indicate that the compounds of the present invention act synergistically or additively with established anti-cancer agents or targeted anticancer inhibitors of PI3K/Akt/mTOR or Jak/STAT pathways in inhibiting the cell growth in the tested cancer cell lines (C: Cytarabine; V: Vosaroxin).

3.17. Determination of a Possible Activity on hERG

(171) The hERG (human ether-A-go-go-related gene) channel corresponds to an important anti-target for potential new drugs since its inhibition may lead to sudden death. In order to establish whether the compounds of the present invention act on hERG, the following experiment was carried out.

(172) The in vitro effects of the compounds indicated in Table 5 on the hERG potassium channel current (a surrogate for I.sub.r, the rapidly activating, delayed rectifier cardiac potassium current) expressed in mammalian cells were evaluated at room temperature using the QPatch HT (Sophion Bioscience A/S, Denmark), an automatic parallel patch clamp system. Each compound indicated in Table 5 was evaluated at 0.1, 1, 3, 10 and 30 M with each concentration tested in a minimum of two cells (n2). The duration of exposure to each compound concentration was 3 minutes. A summary of the results is shown in Table 5. The positive control (E4031) confirmed the sensitivity of the test system to hERG inhibition (98.6% of inhibition at 0.5 M). Generally, compounds displaying an IC50> about 0.5 M are regarded as not acting on hERG and thus as safe.

(173) TABLE-US-00021 TABLE 5 hERG IC50 determination in automated patch clamp assay. Ex. hERG IC50 [M] 1A 1.6 8A 0.77 22L 0.85 22R 1.78 1M 0.52 1N 1.28 2A 3.06 1P 1.88 2C 2.5 2D 1.6 1X 20.42 1Y 16.91 1AA 2.27 22A 10.09 1AB 8.93 1AC 1.86 1AD 1.42 1AE 0.4 1AH 4.99 1AI 0.44 1AL 2.42 1AM 1.46 1AP 1.82 1AQ 1.86 1AR 4.17 1AS 5.77 1AX 1.23 1AY 3.88 1AZ 1.88 22J 11.91 1BI 5.17 1BD 4.34 22AB 2.02 1BM 0.74

(174) As can be derived from the results depicted in Table 5, the compounds of the present invention substantially fail to target hERG and can thus be regarded as safe with respect to the risk of sudden death connected to an hERG-inhibition.

3.18. Determination of a Possible Activity on CYP

(175) In general, drugs should preferably not inhibit cytochrome P450 enzymes such that biotransformation is not negatively influenced. Thus, compounds of the present invention were assayed for their activity on such enzymes (CYP).

(176) The assays for cytochrome P450 inhibition facilitate the identification of drug candidates with lower potential for drug-drug interactions (weak enzymes inhibitors). In vitro experiments were conducted to determine whether a drug inhibits a specific CYP enzyme. The experiments comprised the incubation of the drug with probe substrates for the CYP enzymes, wherein the following recombinant cytochrome P450 isoforms were employed: CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, together with various probe substrates enabling fluorescence detection. The protocol uses a single substrate concentration near the apparent K.sub.m and multiple compound concentrations. An IC.sub.50 is determined as the point where 50% inhibition of enzyme catalytic activity occurs.

(177) The assay was performed in 96-well microtiter plates. The row designations were A through H and the column designations were 1 through 12. This particular experimental design was to perform an IC.sub.50 determination in duplicate rows of 12 wells. Each compound (see Table 6 for tested compounds) was added to the wells in column 1 and serially diluted to the wells in column 8. Wells 9 and 10 were control wells which contained no test compound (therefore no inhibition-full signal is detected.) The wells in columns 11 and 12 were blanks, where STOP solution was added prior to the addition of the enzyme/substrate mix to the NADPH regenerating system (the only signal present in these wells is background noise.) The assay was conducted in a final volume of 0.2 ml per well.

(178) The stock solutions of the tested compounds were prepared in DMSO at 10 mM concentration. Stock solutions of all compounds (tested and control) were prepared 500 times the desired concentration in the assay and diluted 500 times with solution buffer A. The following 8 concentrations of the compounds were used for IC.sub.50 determination: 0.009, 0.027, 0.082, 0.247, 0.741, 2.22, 6.67 and 20 M. After mixing the compounds with solution containing NADPH-cofactors, the mixed plate was preincubated in a 37 C. incubator for at least 10 minutes; next, the fluorescence of compounds using recommended excitation/emission filters was measured in order to eliminate false results originating from autofluorescence of the compounds. In the following step, the enzyme/substrate mix was added to columns 1 through 10 and the plates were incubated at 37 C. for specific times depending on the CYP tested (incubation times ranged from 30 to 45 minutes). After adding STOP SOLUTION to all wells and respective enzyme/substrate mix to the wells in columns 11 and 12, the plate was scanned with a fluorescent plate scanner. The excitation/emission filters used for the specific assays are described in the GenTest Screening Kit instruction manual. The IC.sub.50 is calculated via linear interpolation from the fluorescence data, wherein the following classification was used: Strong inhibition: <1.1 M; Moderate inhibition: 1.1-3.3 M; Mild inhibition: 3.3-10 M; Weak inhibition: >10 M.

(179) TABLE-US-00022 TABLE 6 CYP 3A4 screening results Ex. CYP inhibition 1D Weak 1A Mild 2A Mild 1Q Mild 2H Mild 1AA Moderate 1AE Moderate 1AF Weak 1AH Weak 22G Weak 2I Mild 1AS Mild 1AX Moderate 22I Weak 22J Mild 1BH Weak 1BI Moderate 1BK Weak 1BC Mild 1BD Weak 22AB Mild 1BM Moderate 26A Weak 1M Moderate 2D Moderate 1AH Moderate

(180) The results shown in Table 6 establish that the compounds of the present invention are weak CYP-inhibitors.

(181) Preferred embodiments of the present invention relate to: 1. A compound of formula (I):

(182) ##STR00354##
wherein
X.sup.1 is selected from the group consisting of nitro, cyano, methyl, trifluoromethyl, C(O)T, C(O)OT.sup.4 and S(O).sub.2T.sup.4;
Z and X.sup.2 are each independently selected from the group consisting of F, Cl, Br, I, C.sub.1-3alkyl and trifluoromethyl, with the proviso that Z and X.sup.2 are not both C.sub.1-3alkyl;
X.sup.3 is selected from the group consisting of H, C.sub.1-6alkyl, C.sub.1-6alkenyl, C.sub.1-6alkynyl and a 3- to 6-membered saturated carbocycle or heterocycle, with the proviso that the point of attachment on said heterocycle is carbon, wherein said 3- to 6-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1 and S(O).sub.2N(T.sup.2)(T.sup.3), and wherein said C.sub.1-6alkyl, C.sub.1-6alkenyl and C.sub.1-6alkynyl is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3) and a 3- to 6-membered saturated carbocycle or heterocycle, wherein said 3- to 6-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST, S(O).sub.2T.sup.1 and S(O).sub.2N(T.sup.2)(T.sup.3);
X.sup.4 is either absent or selected from NR.sup.4 and N(R.sup.4)(CH.sub.2);
R.sup.4 is selected from H and C.sub.1-6alkyl;
Y.sup.1 is selected from the group consisting of H, C.sub.1-6alkyl and a 4- to 7-membered saturated or unsaturated aromatic carbocycle or heterocycle, with the proviso that the point of attachment on said heterocycle is carbon if X.sup.4 is NR.sup.4 or N(R.sup.4)(CH.sub.2), wherein said C.sub.1-6alkyl is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3) and a 5- to 6-membered saturated heterocycle, and wherein said 4- to 7-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3), oxo and C.sub.1-3alkyl, wherein said C.sub.1-3alkyl is optionally substituted with one or more substituents independently selected from OT.sup.7, N(T.sup.2)(T.sup.3) and a 6-membered saturated heterocycle;
T.sup.1, T.sup.2 and T.sup.3 are each independently selected from H and C.sub.1-6alkyl optionally substituted with one or more substituents independently selected from F, N(T.sup.5)(T.sup.6), OT.sup.7, ST.sup.7, cyano, C(O)OT.sup.7, C(O)N(T.sup.5)(T.sup.6), OC(O)N(T.sup.5)(T.sup.6), S(O).sub.2T.sup.7, S(O).sub.2OT.sup.8 and S(O).sub.2N(T.sup.5)(T.sup.6);
T.sup.4 is C.sub.1-6alkyl optionally substituted with one or more substituents independently selected from F, N(T.sup.5)(T.sup.6), OT.sup.7, ST.sup.7, cyano, C(O)OT.sup.7, C(O)N(T.sup.5)(T.sup.6), OC(O)N(T.sup.5)(T.sup.6), S(O).sub.2T.sup.8, S(O).sub.2OT.sup.7 and S(O).sub.2N(T.sup.5)(T.sup.6);
T.sup.5, T.sup.6 and T.sup.7 are each independently selected from H and C.sub.1-6alkyl optionally substituted with one or more substituents independently selected from F, amino, hydroxyl, thiol and cyano; and
T.sup.8 is selected from C.sub.1-6alkyl optionally substituted with one or more substituents independently selected from F, amino, hydroxyl, thiol and cyano;
or a pharmaceutically acceptable salt thereof. 2. A compound according to 1, wherein Z and X.sup.2 are each independently selected from the group consisting of F, Cl, Br, I, and trifluoromethyl. 3. A compound according to 1 or 2, wherein X.sup.3 is selected from the group consisting of C.sub.2-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynyl and a 3- to 6-membered saturated carbocycle or heterocycle, with the proviso that the point of attachment on said heterocycle is carbon, wherein said 3- to 6-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1 and S(O).sub.2N(T.sup.2)(T.sup.3), and wherein said C.sub.2-6alkyl, C.sub.2-6alkenyl and C.sub.2-6alkynyl is optionally substituted with one or more substituents independently selected from F, OT, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3) and a 3- to 6-membered saturated carbocycle or heterocycle, wherein said 3- to 6-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT, ST.sup.1, S(O).sub.2T.sup.1 and S(O).sub.2N(T.sup.2)(T.sup.3). 4. A compound according to 1 or 2, wherein X.sup.3 is selected from the group consisting of H, C.sub.1-6alkyl, C.sub.1-6alkenyl, C.sub.1-6alkynyl, wherein said C.sub.1-6alkyl, C.sub.1-6alkenyl and C.sub.1-6alkynyl is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1 and S(O).sub.2N(T.sup.2)(T.sup.3). 5. A compound according to 1 or 2, wherein X.sup.3 is selected from the group consisting of C.sub.1-6alkyl, C.sub.1-6alkenyl, C.sub.1-6alkynyl and a 3- to 6-membered saturated carbocycle or heterocycle, with the proviso that the point of attachment on said heterocycle is carbon, wherein said 3- to 6-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1 and S(O).sub.2N(T.sup.2)(T.sup.3), and wherein said C.sub.1-6alkyl, C.sub.1-6alkenyl and C.sub.1-6alkynyl is substituted with a 3- to 6-membered carbocycle or heterocycle, wherein said 3- to 6-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1 and S(O).sub.2N(T.sup.2)(T.sup.3). 6. A compound according to any one of 1 to 5, wherein X.sup.4 is NR.sup.4 and Y.sup.1 is selected from the group consisting of H and C.sub.1-6alkyl, wherein said C.sub.1-6alkyl is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3). 7. A compound according to any one of 1 to 5, wherein Y.sup.1 is a 4- to 7-membered saturated or unsaturated aromatic carbocycle or heterocycle, with the proviso that the point of attachment on said heterocycle is carbon if X.sup.4 is NR.sup.4 or N(R.sup.4)(CH.sub.2), wherein said 4- to 7-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST.sup.1, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3), oxo and C.sub.1-3alkyl, wherein said C.sub.1-3alkyl is optionally substituted with one or more substituents independently selected from OT.sup.7, N(T.sup.2)(T.sup.3) and a 6-membered saturated heterocycle. 8. A compound according to 7, wherein Y.sup.1 is a 4- to 7-membered saturated carbocycle or heterocycle, with the proviso that the point of attachment on said heterocycle is carbon if X.sup.4 is NR.sup.4 or N(R.sup.4)(CH.sub.2), wherein said 4- to 7-membered carbocycle or heterocycle is optionally substituted with one or more substituents independently selected from F, OT.sup.1, N(T.sup.2)(T.sup.3), C(O)N(T.sup.2)(T.sup.3), C(O)OT.sup.1, ST, S(O).sub.2T.sup.1, S(O).sub.2N(T.sup.2)(T.sup.3), oxo and C.sub.1-3alkyl, wherein said C.sub.1-3alkyl is optionally substituted with one or more substituents independently selected from OT.sup.7, N(T.sup.2)(T.sup.3) and a 6-membered saturated heterocycle. 9. A compound according to 7 or 8, wherein X.sup.4 is absent. 10. A compound according to 1, wherein said compound is selected from the group consisting of: 5,6-dibromo-1-ethyl-4-nitro-2-(piperazin-1-yl)-1H-1,3-benzodiazole; 5,6-dibromo-4-nitro-2-(piperazin-1-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole; 2-[(3R)-3-aminopyrrolidin-1-yl]-5,6-dibromo-1-(propan-2-yl)-1H-1,3-benzodiazole-4-carbonitrile; 2-[(3R)-3-aminopyrrolidin-1-yl]-5,6-dibromo-1-ethyl-1,3-benzodiazole-4-carbonitrile; 5,6-dibromo-2-[(2S)-2-methylpiperazin-1-yl]-4-nitro-1-(propan-2-yl)-1H-1,3-benzodiazole; trans-1-N-[5,6-dibromo-4-nitro-1-(propan-2-yl)-1H-1,3-benzodiazol-2-yl]cyclohexane-1,4-diamine; 5,6-dibromo-1-cyclopentyl-4-nitro-2-(piperazin-1-yl)-1H-1,3-benzodiazole hydrochloride; 5,6-dibromo-4-nitro-2-(piperazin-1-yl)-1-propyl-1H-1,3-benzodiazole; 5,6-dibromo-1-(2-methylpropyl)-4-nitro-2-(piperazin-1-yl)-1H-1,3-benzodiazole; 5,6-dibromo-1-(cyclopropylmethyl)-4-nitro-2-(piperazin-1-yl)-1H-1,3-benzodiazole; (3S)-1-(5,6-dibromo-1-ethyl-4-nitro-1H-1,3-benzodiazol-2-yl)piperidin-3-amine; (3S)-1-[5,6-dibromo-4-nitro-1-(propan-2-yl)-1H-1,3-benzodiazol-2-yl]piperidin-3-amine; (3S)-1-[5,6-dibromo-4-nitro-1-(propan-2-yl)-1H-1,3-benzodiazol-2-yl]pyrrolidin-3-amine; (3R)-1-[5,6-dibromo-4-nitro-1-(propan-2-yl)-1H-1,3-benzodiazol-2-yl]pyrrolidin-3-amine; 5,6-dibromo-4-nitro-1-(propan-2-yl)-N-[(3S)-pyrrolidin-3-yl]-1H-1,3-benzodiazol-2-amine hydrochloride; 2-[(3S)-3-aminopiperidin-1-yl]-5,6-dibromo-1-ethyl-1,3-benzodiazole-4-carbonitrile hydrochloride; 5,6-dibromo-4-nitro-N-[(3S)-piperidin-3-yl]-1-(propan-2-yl)-1H-1,3-benzodiazol-2-amine hydrochloride; and 5,6-dibromo-1-(2-methylpropyl)-4-nitro-2-(piperazin-1-yl)-1H-1,3-benzodiazole hydrochloride. 11. A compound according to any one of 1 to 10, wherein the pharmaceutically acceptable salt is selected from the group consisting of the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate. 12. A pharmaceutical composition comprising a compound according to any one of 1 to 11. 13. A pharmaceutical composition according to 12 for use in the treatment of a disease selected from the group consisting of cancer, an autoimmune disease and an inflammatory disease. 14. A pharmaceutical composition according to 12 or 13 for use in the treatment of a disease selected from the group consisting of leukemias including acute lymphoblastic leukemia, acute myelogenous leukemia and chronic lymphocytic leukemia, lymphoma, myeloma, myeloproliferative disorder, allograft rejection, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythamatosus, Alzheimer disease and Down syndrome. 15. Method for modulating or regulating and preferably inhibiting serine/threonine or tyrosine kinases, preferably selected from the group consisting of PIM1-3, FLT3 and DYRK1A and more preferably selected from the group consisting of PIM1-3 and DYRK1A, wherein said serine/threonine or tyrosine kinases are exposed to at least one compound of formula (I) according to any one of 1 to 11, wherein said method is preferably performed outside the human or animal body. 16. Use of a compound of formula (I) according to any one of 1 to 11 as serine/threonine or tyrosine kinase modulating and preferably inhibiting agent, wherein said kinase is preferably selected from the group consisting of PIM1-3, FLT3 and DYRK1A and more preferably selected from the group consisting of PIM1-3 and DYRK1A.