WO2015049325A1 - Therapeutic inhibitors of cdk8 and uses thereof - Google Patents

Abstract

The present invention relates to compounds formula (I) and to salts thereof, wherein R¹-R⁴ have any of the values defined in the specification, and compositions and uses thereof. The compounds are useful as inhibitors of CDK8. Also included are pharmaceutically acceptable compositions comprising the compounds of the present invention and methods of using said compositions in the treatment of various disorders.

Description

THERAPEUTIC INHIBITORS OF CDK8 AND USES THEREOF

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds useful as inhibitors of CDK8.

BACKGROUND OF THE INVENTION

CDK8 is a component of the mediator complex. The mediator complex plays a central role in transcription by coupling sequence- specific transcription factors with the transcriptional machinery. CDK8 forms the CDK8 module of the complex along with Cyclin C, MED12, and MED13. It phosphorylates the C-terminal domain of RNA polymerase II, serine 10 of Histone H3, and serine 206 of Smadl in addition to a number of other substrates. It modulates transcriptional pathways including beta-catenin, Notch, p53, serum response factor, and SMAD. It also acts indirectly on the beta-catenin pathway by phosphorylating E2F1 and thereby reversing its repression of beta-catenin activity.⁴' ⁵

CDK8 is an oncogene.⁶'⁷ It is amplified and overexpressed in colon cancers.

Additionally, it is able to transform NIH3T3 cells as evidenced by loss of contact inhibition, anchorage independent growth, and growth of tumors in vivo. Notably, transformation does not occur with kinase dead CDK8, so it appears to be dependent upon kinase activity. Knockdown of CDK8 in cell lines with copy number gain inhibits cell proliferation. Additionally, tumor growth inhibition was observed in murine xenograft models with inducible CDK8 knockdown.⁸ One proposed mechanism of CDK8-mediated inhibition in colon cancer is down-regulation of the beta-catenin pathway in cancers with high levels of CDK8. However, the knockdown results do not enable discrimination of the importance of the scaffolding and kinase functions of CDK8.

CDK8 and its partner Cyclin C are dysregulated in human cancers.⁹ Retrospective analyses of human colon tumor samples show that expression of CDK8 correlates with beta- catenin activation and poor prognosis.^{10 11} CDK8 was also found to induce an embryonic stem cell signature that is enriched in colon tumors with poor differentiation and correlates with poor prognosis.⁸ Additional cell line, murine xenograft, and human tumor tissue studies have also linked CDK8 to melanoma.¹³ The therapeutic hypothesis underlying this work is that small molecule inhibitors of CDK8 will inhibit the growth of the subset of tumors that exhibit CDK8 amplification and/or overexpression. Potential indications include colon cancer and melanoma. There is currently a need for compounds that inhibit of CDK8 for treating hyperproliferative diseases

SUMMARY OF THE INVENTION

One aspect includes a compound of formula (I):

or a salt thereof, wherein:

1 is selected from:

and wherein R¹ is independently optionally substituted by 1, 2 or 3 R^x groups;

R 2" and R 3^J are independently H, Q-nalkyl, C₂_₁₂alkenyl, C₂_₁₂alkynyl, carbocyclyl, aryl, heteroaryl, heterocyclyl, halo, -OR^a, -SR^a, -N(R^a)₂, -CN, -N0₂, -C(0)R^a, -C0₂R^a, -C(0)N(R^a)₂, -C(0)SR^a, -C(0)C(0)R^a, -C(0)CH₂C(0)R^a, -C(S)N(R^a)₂, -C(S)OR^a, -S(0)R^a, -S0₂R^a, -S0₂N(R^a)₂, -N(R^a)C(0)R^a, -N(R^a)C(0)N(R^a)₂, -N(R^a)S0₂ R^a,

-N(R^a)S0₂N(R^a)₂, -N(R^a)N(R^a)₂, -N(R^a)C(=N(R^a))N(R^a)₂, -C(=N)N(R^a)₂, -C=NOR^a, -C(=N(R^a))N(R^a)₂, -OC(0)R^a, or -OC(0)N(R^a)₂, wherein each d_i₂alkyl, C₂_i₂alkenyl,

C₂_₁₂alkynyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl of R 2 and R 3 are each independently optionally substituted with one or more groups R^x; or R¹ and R² taken together with the atoms to which they are attached form a 4, 5, 6, 7, or 8 membered carbocyclyl, heterocyclyl, heteroaryl or aryl, which carbocyclyl, heterocyclyl, heteroaryl and aryl is optionally substituted with one or more groups R^x;

each R^a and R^b are independently H, Ci^alkyl, C₂_₆alkenyl, C₂_₆alkynyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl, wherein each Ci^alkyl, C₃_₆alkenyl, C₃_₆alkynyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl is independently optionally substituted with one or more groups R^x; or R^a and R^b are taken together with the nitrogen to which they are attached to form a 3-6 membered heterocyle optionally substituted by oxo, halo, or C^alkyl optionally subsituted by oxo or halo; each R^v is independently hydrogen, C^alkyl, C₂_₆alkenyl, C₂_₆alkynyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl, wherein each Ci^alkyl, C₂_₆alkenyl, C₂_₆alkynyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl is optionally substituted with one or more groups independently selected from oxo, halo, amino, hydroxyl, and C^Ce alkyl that is optionally substituted with one or more groups independently selected from oxo and halo; or two R^v are taken together with the nitrogen to which they are attached to form a 3-6 membered heterocyclyl that is optionally substituted with one or more groups independently selected from oxo, halo and C^alkyl that is optionally substituted with one or more groups independently selected from oxo and halo; and

each R^x is independently oxo, Ci^alkyl, C₂_₆alkenyl, C₂_₆alkynyl, Ci^haloalkyl,

carbocyclyl, aryl, heteroaryl, heterocyclyl, -F, -CI, -Br, -I, -N0₂, -N(R^V)₂, -CN, -C(O)- N(R^V)₂, -S(0)-N(R^v)₂, -S(0)₂-N(R^v)₂, -0-R^v, -S-R^v, -0-C(0)-R^v, -0-C(0)-0-R^v, -C(O)- R^v, -C(0)-0-R^v, -S(0)-R^v, -S(0)₂-R^v, -0-C(0)-N(R^v)₂, -N(R^v)-C(0)-OR^v, -N(R^v)-C(0)- N(R^V)₂, -S(0)₂-N(R^v)₂, -N(R^v)-C(0)-R^v, -N(R^v)-S(0)-R^v, -N(R^v)-S(0)₂-R^v, -N(R^v)-S(0)- N(R^V)₂, or -N(R^v)-S(0)₂-N(R^v)₂, wherein any Ci^alkyl, C₂_₆alkenyl, C₂_₆alkynyl, Ci_ 6haloalkyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl is independently optionally substituted with one or more groups selected from oxo, halo, -N0₂, -N(R^V)₂, -CN,-C(0)- N(R^V)₂, -S(0)-N(R^v)₂, -S(0)₂-N(R^v)₂, -0-R^v, -S-R^v, -0-C(0)-R^v, -C(0)-R^v, -C(0)-0-R^v, - S(0)-R^v, -S(0)₂-R^v, -C(0)-N(R^v)₂, -S(0)₂-N(R^v)₂, -N(R^v)-C(0)-R^v, -N(R^v)-S(0)-R^v - N(R^v)-S(0)₂-R^v and C^alkyl that is optionally substituted with one or more groups independently selected from oxo and halo; and

wherein when R² is F and R³ is hydrogen, then R^x is other than methyl, ethyl, trifluoromethyl or 2-fluorophenyl.

Another aspect includes a pharmaceutical composition, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant, carrier, or vehicle.

Another aspect includes a method of treating a disease associated with CDK8 activity, comprising administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, to a patient in need thereof.

Another aspect includes the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in therapy. Another aspect includes the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the treatment of a hyperproliferative disorder, in one example, cancer. Another aspect includes the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease associated with CDK8 activity.

Another aspect includes compounds for the study of kinases, such as CDK8, the study of intracellular signal transduction pathways mediated by such CDK8, and the comparative evaluation of modulators of CDK8.

Another aspect includes a process for preparing a compound of formula I or a salt thereof.

DETAILED DESCRIPTION

Compounds and Definitions

Definitions and terms are described in more detail below. Chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed.

Unless otherwise stated, compounds of formula (I) include enantiomeric,

diastereomeric and geometric (or conformational)) isomeric forms of a given structure. For example, the R and S configurations for each asymmetric center, Z and E double bond isomers, Z and E conformational isomers, , single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures are included. Unless otherwise stated, all tautomeric forms of structures depicted herein are included. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds of formula I, wherein the independent replacement or endrichment of one or more: hydrogen by deuterium or tritium, carbon by ¹³C- or ¹⁴C carbon, nitrogen by a

15 N nitrogen, sulfur by a 33 S, 34 S or 36 S sulfur, or oxygen by a 17 O or 18 O oxygen, are included. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents.

Where a particular enantiomer is described, it may, in certain embodiments be provided substantially free of the corresponding enantiomer, and may also be referred to as "optically enriched." "Optically-enriched," as used herein, means that the mixture of enantiomers is made up of a significantly greater proportion of one enantiomer, and may be described by enantiomeric excess (ee %). In certain embodiments, the mixture of enantiomers is made up of at least about 90% by weight of a given enantiomer (about 90% ee). In other embodiments, the mixture of enantiomers is made up of at least about 95%, 98% or 99% by weight of a given enantiomer (about 95%, 98% or 99% ee). Enantiomers and diastereomers may be isolated from racemic mixtures by any method known to those skilled in the art, including recrystallization from solvents in which one stereoisomer is more soluble than the other, chiral high pressure liquid chromatography (HPLC), supercritical fluid chromatography (SFC), the formation and crystallization of chiral salts, which are then separated by any of the above methods, or prepared by asymmetric syntheses and optionally further enriched. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).

The term "heteroatom" means any atom independently selected from an atom other than carbon or hydrogen, for example, one or more of oxygen, sulfur, nitrogen, phosphorus or silicon (including any oxidized form of nitrogen, sulfur, phosphorus or silicon; and the quaternized form of any nitrogen).

The terms "halo" and "halogen" as used herein refer to an atom selected from fluorine (fluoro, -F), chlorine (chloro, -CI), bromine (bromo, -Br) and iodine (iodo, -I). Particular examples of halo are fluoro and chloro.

The term "unsaturated", as used herein, means that a moiety has one or more units of unsaturation.

The term "carbocyclyl" used alone or as part of a larger moiety, refers to a saturated, partially unsaturated, or aromatic ring system having 3 to 20 carbon atoms. In one embodiment, carbocyclyl includes 3 to 12 carbon atoms (C₃-C₁₂). In another embodiment, carbocyclyl includes C₃-C₈, rCio or Cs-Cio- In other embodiment, carbocyclyl, as a monocycle, includes C₃-C₈, C₃-C₆ or C5-C₆. In another embodiment, carbocyclyl, as a bicycle, includes C₇-C₁₂. In another embodiment, carbocyclyl, as a spiro system, includes C₅-C₁₂. Examples of monocyclic carbocyclyls include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-l-enyl, l-cyclopent-2-enyl, l-cyclopent-3-enyl, cyclohexyl,

perdeuteriocyclohexyl, 1-cyclohex-l-enyl, l-cyclohex-2-enyl, l-cyclohex-3-enyl,

cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, phenyl, and cyclododecyl; bicyclic carbocyclyls having 7 to 12 ring atoms include [4,3], [4,4], [4,5], [5,5], [5,6] or [6,6] ring systems, for example bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, naphthalene, and bicyclo[3.2.2]nonane; and spiro carbocyclyls include spiro[2.2]pentane, spiro[2.3]hexane, spiro[2.4]heptane, spiro[2.5]octane and spiro[4.5]decane, each of which are independently optionally substituted with one or more groups described herein. The term carbocyclyl includes aryl ring systems as defined herein.

The term "alkyl," as used herein, refers to a saturated linear or branched-chain monovalent hydrocarbon radical. In one embodiment, the alkyl radical is one to eighteen carbon atoms (C C^). In other embodiments, the alkyl radical is Co-C₆, C₀-C₅, C₀-C3, Q- C₁₂, Q-Cio_, Q-Cg, C -C , Q-C₅, Q-C4 or Ci-C^. Co alkyl refers to a bond. Examples of alkyl groups include methyl (Me, -CH₃), ethyl (Et, -CH₂CH₃), 1 -propyl (n-Pr, n-propyl, - CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, -CH(CH₃)₂), 1 -butyl (n-Bu, n-butyl, - CH₂CH₂CH₂CH₃), 2-methyl- l -propyl (i-Bu, i-butyl, -CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, - CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH₃)₃), 1-pentyl (n-pentyl, - CH₂CH₂CH₂CH₂CH₃), 2-pentyl (-CH(CH₃)CH₂CH₂CH₃), 3-pentyl (-CH(CH₂CH₃)₂), 2- methyl-2-butyl (-C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl (-CH(CH₃)CH(CH₃)₂), 3-methyl-l- butyl (-CH₂CH₂CH(CH₃)₂), 2-methyl- l -butyl (-CH₂CH(CH₃)CH₂CH₃), 1-hexyl (- CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl (-CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (- CH(CH₂CH₃)(CH₂CH₂CH₃)), 2-methyl-2-pentyl (-C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl (-CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (-CH(CH₃)CH₂CH(CH₃)₂), 3-methyl-3- pentyl (-C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl (-CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2- butyl (-C(CH₃)₂CH(CH₃)₂), 3,3-dimethyl-2-butyl (-CH(CH₃)C(CH₃)₃, heptyl, octyl, nonyl, decyl, undecyl and dodecyl. Particular examples of C_1-12alkyl are C_1-6alkyl. Particular exaples of Ci-₆alkyl are methyl and ethyl.

The term "alkenyl," as used herein, denotes a linear or branched-chain monovalent hydrocarbon radical with at least one carbon-carbon double bond. An alkenyl includes radicals having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations. In one example, the alkenyl radical is two to eighteen carbon atoms (C₂-Ci₈). In other examples, the alkenyl radical is C₂-C₁₂, C₂-Cio_, C₂-C₈, C₂-C₆ or C₂-C3. Examples include, but are not limited to, ethenyl or vinyl (-CH=CH₂), prop-l-enyl (-CH=CHCH₃), prop-2-enyl (- CH₂CH=CH₂), 2-methylprop-l-enyl, but-l-enyl, but-2-enyl, but-3-enyl, buta- l,3-dienyl, 2- methylbuta- l,3-diene, hex-l-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl and hexa-l,3-dienyl.

The term "alkynyl," as used herein, refers to a linear or branched monovalent hydrocarbon radical with at least one carbon-carbon, triple bond. In one example, the alkynyl radical is two to eighteen carbon atoms (C₂-Ci₈). In other examples, the alkynyl radical is C₂- C₁₂, C₂-Cio_, C₂-C₈, C₂-C₆ or C₂-C₃. Examples include, but are not limited to, ethynyl (-C≡CH), prop-l-ynyl (-C≡CCH₃), prop-2-ynyl (propargyl, -CH₂C≡CH), but-l-ynyl, but-2- ynyl and but-3-ynyl.

The term "alkoxy" refers to a linear or branched monovalent radical represented by the formula -OR in which R is alkyl, alkenyl, alkynyl or carbocycyl. Alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, mono-, di- and tri-fluoromethoxy and cyclopropoxy. Particular example of alkoxy is methoxy.

The term "haloalkyl," as used herein, refers to an alkyl as defined herein that is substituted with one or more (e.g. 1, 2, 3, or 4) halo groups. Particular examples of haloalkyl are trifluoromethyl and difiuoromethyl.

The term "aryl" used alone or as part of a larger moiety as in "arylalkyl",

"arylalkoxy", or "aryloxyalkyl", refers to a monocyclic, bicyclic or tricyclic, carbon ring system, that includes fused rings, wherein at least one ring in the system is aromatic. The term "aryl" may be used interchangeably with the term "aryl ring". In one embodiment, aryl includes groups having 6-18 carbon atoms. In another embodiment, aryl includes groups having 6-10 carbon atoms. Examples of aryl groups include phenyl, naphthyl, anthracyl, biphenyl, phenanthrenyl, naphthacenyl, 1,2,3,4-tetrahydronaphthalenyl, lH-indenyl, 2,3- dihydro-lH-indenyl, and the like, which may be substituted or independently substituted byone or more substituents described herein. A particular aryl is phenyl. In another embodiment aryl includes an aryl ring fused to one or more carbocyclic rings, such as indanyl, phthalimidyl, naphthimidyl, phenantriidinyl, or tetrahydronaphthyl, and the like, where the radical or point of attachment is on an aromatic ring.

The term "heteroaryl" used alone or as part of a larger moiety, e.g., "heteroarylalkyl", or "heteroarylalkoxy", refers to a monocyclic, bicyclic or tricyclic ring system having 5 to 14 ring atoms, wherein at least one ring is aromatic and contains at least one heteroatom. In one embodiment, heteroaryl includes 4-6 membered monocyclic aromatic groups where one or more ring atoms is nitrogen, sulfur or oxygen that is independently optionally substituted. In another embodiment, heteroaryl includes 5-6 membered monocyclic aromatic groups where one or more ring atoms is nitrogen, sulfur or oxygen that is independently optionally substituted. Example heteroaryl groups include thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, tetrazolo[l,5-b]pyridazinyl, imidazol[l,2-a]pyrimidinyl, purinyl, benzoxazolyl, benzofuryl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoimidazolyl, indolyl, l,3-thiazol-2-yl, l,3,4-triazol-5-yl, l,3-oxazol-2-yl, l,3,4-oxadiazol-5-yl, l,2,4-oxadiazol-5-yl, 1,3,4- thiadiazol-5-yl, lH-tetrazol-5-yl, l,2,3-triazol-5-yl, and pyrid-2-yl N-oxide,. The terms "heteroaryl" also includes groups in which a heteroaryl is fused to one or more aryl, carbocyclyl, or heterocyclyl rings, where the radical or point of attachment is on the heteroaryl ring. Nonlimiting examples include indolyl, isoindolyl, benzothienyl,

benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,

tetrahydroisoquinolinyl and pyrido[2,3-b]- l,4-oxazin-3(4H)-one. A heteroaryl group may be mono-, bi- or tri-cyclic.

As used herein, the term "heterocyclyl" refers to a "carbocyclyl" as defined herein, wherein one or more (e.g. 1, 2, 3, or 4) carbon atoms have been replaced with a heteroatom (e.g. O, N, or S). A heterocyclyl can optionally be substituted with one or more substituents independently selected from those defined herein.

In one example, heterocyclyl includes 3-12 ring atoms and includes monocycles, bicycles, tricycles and spiro ring systems, wherein the ring atoms are carbon, and one to five ring atoms is a heteroatom selected from nitrogen, sulfur or oxygen, which is independently optionally substituted by one or more groups. In one example, heterocyclyl includes 1 to 4 heteroatoms. In another example, heterocyclyl includes 3- to 7-membered monocycles having one or more heteroatoms selected from nitrogen, sulfur or oxygen. In another example, heterocyclyl includes 4- to 6-membered monocycles having one or more heteroatoms selected from nitrogen, sulfur or oxygen. In another example, heterocyclyl includes 3-membered monocycles. In another example, heterocyclyl includes 4-membered monocycles. In another example, heterocyclyl includes 5-6-membered monocycles. In one example, the heterocyclyl group includes 0 to 3 double bonds. Any nitrogen or sulfur heteroatom may optionally be oxidized (e.g. NO, SO, S0₂), and any nitrogen heteroatom may optionally be quaternized (e.g. [NR₄]⁺Cr, [NR₄]⁺OH^~). Example heterocyclyls include oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 1,2-dithietanyl, 1,3-dithietanyl, pyrrolidinyl, dihydro-lH-pyrrolyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothienyl, tetrahydrothienyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 1, 1-dioxo-thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl, oxazinanyl, thiazinanyl, thioxanyl, homopiperazinyl,

homopiperidinyl, azepanyl, oxepanyl, thiepanyl, oxazepinyl, oxazepanyl, diazepanyl, 1,4- diazepanyl, diazepinyl, thiazepinyl, thiazepanyl, tetrahydrothiopyranyl, oxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,1-dioxoisothiazolidinonyl, oxazolidinonyl, imidazolidinonyl, 4,5,6,7-tetrahydro[2H]indazolyl, tetrahydrobenzoimidazolyl, 4,5,6,7- tetrahydrobenzo[d]imidazolyl, 1 ,6-dihydroimidazol[4,5-d]pyrrolo[2,3-b]pyridinyl, thiazinyl, oxazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl, 1- pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, thiapyranyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrimidinonyl, pyrimidindionyl, pyrimidin-2,4-dionyl, piperazinonyl, piperazindionyl,

pyrazolidinylimidazolinyl, 3-azabicyclo[3.1.0]hexanyl, 3,6-diazabicyclo[3.1.1]heptanyl, 6- azabicyclo[3.1. l]heptanyl, 3-azabicyclo[3.1. l]heptanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 2-azabicyclo[3.2.1]octanyl, 8-azabicyclo[3.2.1]octanyl, 2- azabicyclo[2.2.2]octanyl, 8-azabicyclo[2.2.2]octanyl, 7-oxabicyclo[2.2. l]heptane, azaspiro[3.5]nonanyl, azaspiro[2.5]octanyl, azaspiro[4.5]decanyl, l-azaspiro[4.5]decan-2- only, azaspiro[5.5]undecanyl, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindazolyl, 1,1-dioxohexahydrothiopyranyl. Examples of 5-membered

heterocyclyls containing a sulfur or oxygen atom and one to three nitrogen atoms are thiazolyl, including thiazol-2-yl and thiazol-2-yl N-oxide, thiadiazolyl, including 1,3,4- thiadiazol-5-yl and l,2,4-thiadiazol-5-yl, oxazolyl, for example oxazol-2-yl, and oxadiazolyl, such as l,3,4-oxadiazol-5-yl, and l,2,4-oxadiazol-5-yl. Example 5-membered ring heterocyclyls containing 2 to 4 nitrogen atoms include imidazolyl, such as imidazol-2-yl; triazolyl, such as l,3,4-triazol-5-yl; l,2,3-triazol-5-yl, l,2,4-triazol-5-yl, and tetrazolyl, such as lH-tetrazol-5-yl. Example benzo-fused 5-membered heterocyclyls are benzoxazol-2-yl, benzthiazol-2-yl and benzimidazol-2-yl. Example 6-membered heterocyclyls contain one to three nitrogen atoms and optionally a sulfur or oxygen atom, for example pyridyl, such as pyrid-2-yl, pyrid-3-yl, and pyrid-4-yl; pyrimidyl, such as pyrimid-2-yl and pyrimid-4-yl; triazinyl, such as l,3,4-triazin-2-yl and l,3,5-triazin-4-yl; pyridazinyl, in particular pyridazin- 3-yl, and pyrazinyl. The pyridine N-oxides and pyridazine N-oxides and the pyridyl, pyrimid-2-yl, pyrimid-4-yl, pyridazinyl and the l,3,4-triazin-2-yl groups, are other example heterocyclyl groups. Particular examples for heterocyclyl are azetidinyl, pyrrolidinyl, piperazinyl and morpholinyl, more particularly azetidin-l-yl, pyrrolidin-l-yl, piperazin-l-yl and morpholin-4-yl. As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond between ring atoms but the ring moiety is not aromatic.

As used herein, the term "inhibitor" refers to a compound that binds to and inhibits a CDK8 enzyme with measurable biochemical affinity and activity. In certain embodiments, an inhibitor has a biochemical IC₅₀ and/or binding constant of less about 50 μΜ, less than about 1 μΜ, less than about 500 nM, less than about 100 nM, or less than about 10 nM.

"Pharmaceutically acceptable salts" include both acid and base addition salts.

"Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid and the like, and organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, salicyclic acid and the like.

"Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly base addition salts are the ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from

pharmaceutically acceptable organic nontoxic bases includes salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, tromethamine, dicyclohexylamine, choline, and caffeine.

The term "tautomer" or "tautomeric form" refers to structural isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include

interconversions by reorganization of some of the bonding electrons.

A "solvate" refers to an association or complex of one or more solvent molecules and a compound of the present invention. Examples of solvents include water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine. The term "hydrate" refers to the complex where the solvent molecule is water.

"Therapeutically effective amount" refers to an amount of a compound of the present invention that (i) treats the particular disease, condition or disorder, (ii) attenuates, ameliorates or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition or disorder described herein. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR). In the case of immunological disorders, the therapeutic effective amount is an amount sufficient to decrease or alleviate an allergic disorder, the symptoms of an autoimmune and/or inflammatory disease, or the symptoms of an acute inflammatory reaction (e.g. asthma). In some embodiments, a therapeutically effective amount is an amount of a chemical entity described herein sufficient to significantly decrease the activity or number of drug tolerant or drug tolerant persisting cancer cells.

"Treatment" (and variations such as "treat" or "treating") refers to clinical

intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology.

Desirable effects of treatment include one or more of preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, stabilized (i.e., not worsening) state of disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, prolonging survival as compared to expected survival if not receiving treatment and remission or improved prognosis. In certain embodiments, a compound of formula I is used to delay development of a disease or disorder or to slow the progression of a disease or disorder. Those individuals in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder, (for example, through a genetic mutation or abberent expression of a gene or protein) or those in which the condition or disorder is to be prevented.

Exemplary Values

One embodiment includes a compound of formula (I):

or a salt thereof, wherein:

1 is selected from:

and wherein R¹ is independently optionally substituted by 1, 2 or 3 R^x groups;

R 2" and R 3^J are independently H, Q-^alkyl, C₂-i₂alkenyl, C₂-i₂alkynyl, carbocyclyl, aryl, heteroaryl, heterocyclyl, halo, -OR^a, -SR^a, -N(R^a)₂, -CN, -N0₂, -C(0)R^a, -C0₂R^a, -C(0)N(R^a)₂, -C(0)SR^a, -C(0)C(0)R^a, -C(0)CH₂C(0)R^a, -C(S)N(R^a)₂, -C(S)OR^a, -S(0)R^a, -S0₂R^a, -S0₂N(R^a)₂, -N(R^a)C(0)R^a, -N(R^a)C(0)N(R^a)₂, -N(R^a)S0₂ R^a,

-N(R^a)S0₂N(R^a)₂, -N(R^a)N(R^a)₂, -N(R^a)C(=N(R^a))N(R^a)₂, -C(=N)N(R^a)₂, -C=NOR^a, -C(=N(R^a))N(R^a)₂, -OC(0)R^a, or -OC(0)N(R^a)₂, wherein each Ci_i₂alk l, C₂_₁₂alkenyl,

C_{2 12}alkynyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl of R 2 and R 3 are each independently optionally substituted with one or more groups R^x; or R¹ and R² taken together with the atoms to which they are attached form a 4, 5, 6, 7, or 8 membered carbocyclyl, heterocyclyl, heteroaryl or aryl, which carbocyclyl, heterocyclyl, heteroaryl and aryl is optionally substituted with one or more groups R^x;

each R^a and R^b are independently H, C^a cyl, C₂-₆alkenyl, C₂-₆alkynyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl, wherein each C^ancyl, C₃_₆alkenyl, C₃_₆alkynyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl is independently optionally substituted with one or more groups R^x; or R^a and R^b are taken together with the nitrogen to which they are attached to form a 3-6 membered heterocyle optionally substituted by oxo, halo, or C^alkyl optionally subsituted by oxo or halo;

each R^v is independently hydrogen, C^alkyl, C₂_₆alkenyl, C₂_₆alkynyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl, wherein each C^ancyl, C₂_₆alkenyl, C₂_₆alkynyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl is optionally substituted with one or more groups independently selected from oxo, halo, amino, hydroxyl, and C C₆ alkyl that is optionally substituted with one or more groups independently selected from oxo and halo; or two R^v are taken together with the nitrogen to which they are attached to form a 3-6 membered heterocyclyl that is optionally substituted with one or more groups independently selected from oxo, halo and Ci^alkyl that is optionally substituted with one or more groups independently selected from oxo and halo; and

each R^x is independently oxo, C^a cyl, C₂_₆alkenyl, C₂_₆alkynyl, Ci-ehaloalkyl,

carbocyclyl, aryl, heteroaryl, heterocyclyl, -F, -CI, -Br, -I, -N0₂, -N(R^V)₂, -CN, -C(O)- N(R^V)₂, -S(0)-N(R^v)₂, -S(0)₂-N(R^v)₂, -0-R^v, -S-R^v, -0-C(0)-R^v, -0-C(0)-0-R^v, -C(O)- R^v, -C(0)-0-R^v, -S(0)-R^v, -S(0)₂-R^v, -0-C(0)-N(R^v)₂, -N(R^v)-C(0)-OR^v, -N(R^v)-C(0)- N(R^V)₂, -S(0)₂-N(R^v)₂, -N(R^v)-C(0)-R^v, -N(R^v)-S(0)-R^v, -N(R^v)-S(0)₂-R^v, -N(R^v)-S(0)- N(R^V)₂, or -N(R^v)-S(0)₂-N(R^v)₂, wherein any C^alkyl, C₂_₆alkenyl, C₂_₆alkynyl, Ci_ 6haloalkyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl is independently optionally substituted with one or more groups selected from oxo, halo, -N0₂, -N(R^V)₂, -CN,-C(0)- N(R^V)₂, -S(0)-N(R^v)₂, -S(0)₂-N(R^v)₂, -0-R^v, -S-R^v, -0-C(0)-R^v, -C(0)-R^v, -C(0)-0-R^v, - S(0)-R^v, -S(0)₂-R^v, -C(0)-N(R^v)₂, -S(0)₂-N(R^v)₂, -N(R^v)-C(0)-R^v, -N(R^v)-S(0)-R^v - N(R^v)-S(0)₂-R^v and C^alkyl that is optionally substituted with one or more groups independently selected from oxo and halo; and

wherein when R² is F and R³ is hydrogen, then R^x is other than methyl, ethyl, trifluoromethyl or 2-fluorophenyl.

A particular embodiment relates to the compound of formula (I) or a salt thereof, wherein R¹ is selected from:

R² and R³ are independently H, Ci_₁₂alkyl, halo, or -OR^a, wherein each Ci_₁₂alkyl of R² and R³ are each independently optionally substituted with one or more groups R^x; each R^a and R^b are independently H, C^alkyl, wherein each Ci^alkyl is independently optionally substituted with one or more groups R^x;

each R^v is independently C^a cyl; and

each R^x is independently heterocyclyl, -F, or -N(R^V)₂, wherein any heterocyclyl is

independently optionally substituted with one or more groups selected from C^aUcyl; and

wherein when R² is F and R³ is hydrogen, then R^x is other than methyl, ethyl, trifluoromethyl or 2-fluorophenyl.

In certain embodiments, R 2 and R 3 are independently halo, trifluoromethyl, C^aUcyl or C^alkoxy, wherein said alkyl and alkoxy are independently optionally substituted by - N(R^V)₂ or heterocyclyl; wherein said heterocyclyl is optionally substituted by oxo, halo or Q_ ₆alkyl optionally substituted by halo or oxo.

In certain embodiments, R is hydrogen, chloro, methyl, (4-methylpiperazin-l- yl)methyl, trifluoromethoxyl, difluoromethoxyl, 4-azetidin-l-ylmethyl, morpholin-4- ylmethyl, dimethylaminomethyl, pyrrolidin-l-ylmethyl or 4-ethylpiperizin-l-ylmethyl.

In certain embodiments, R is hydrogen, trifluoromethyl, fluoro or chloro.

In certain embodiments, each R^a and R^b are independently hydrogen or C_halk !.

In certain embodiments, R^a is hydrogen.

In certain embodiments, R^b is methyl.

In certain embodiments, R^v is hydrogen or C^alkyl.

In certain embodiments, R^v is methyl.

In certain embodiments, R is hydrogen, chloro, methyl, (4-methylpiperazin-l-yl)methyl, trifluoromethoxyl, difluoromethoxyl, 4-azetidin-l-ylmethyl, morpholin-4-ylmethyl, dimethylaminomethyl, pyrrolidin-l-ylmethyl or 4-ethylpiperizin-l-ylmethyl; R is hydrogen, trifluoromethyl or chloro; R^a and R^b are independently hydrogen or methyl.

Another embodiment includes a compound of the following formula:

or a salt thereof, wherein R 1 , R2 , and R 3 are as defined for any of the embodiments for a comopund of formula I.

Another embodiment includes a compound selected from the group consisting of: l-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl]-3-[3- (trifluoromethyl)phenyl]urea;

l-[4-chloro-3-(trifluoromethyl)phenyl]-3-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6- yl)pyrrolidin-3-yl]urea;

l-(3-chlorophenyl)-3-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl]urea; l-[(3R)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl]-3-[3-

(trifluoromethyl)phenyl]urea;

l-[4-chloro-3-(trifluoromethyl)phenyl]-3-[(3R)-l-([l,2,4]triazolo[4,3-b]pyridazin-6- yl)pyrrolidin-3-yl]urea;

l-(3-chlorophenyl)-3-[(3R)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl]urea; (S)-l-(l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl)-3-(4-((4-ethylpiperazin-l- yl)methyl)-3-(trifluoromethyl)phenyl)urea;

l-[(3S)-l-(2-aminopyrimidin-4-yl)pyrrolidin-3-yl]-3-(3-chloro-4-methyl-phenyl)urea;

l-[(S)-l-(2-Amino-pyrimidin-4-yl)-pyrrolidin-3-yl]-3-(4-chloro-3-trifluoromethyl-phenyl)- urea;

l-[4-chloro-3-(trifluoromethyl)phenyl]-3-[(3S)-l-[2-(methylamino)pyrimidin-4-yl]pyrrolidin- 3-yl]urea;

l-(3-chloro-4-methyl-phenyl)-3-[(3S)-l-[2-(methylamino)pyrimidin-4-yl]pyrrolidin-3- yl]urea;

l-[(3S)-l-[2-(methylamino)pyrimidin-4-yl]pyrrolidin-3-yl]-3-[4- (trifluoromethoxy)phenyl]urea;

l-[4-(difluoromethoxy)phenyl]-3-[(3S)-l-[2-(methylamino)pyrimidin-4-yl]pyrrolidin-3- yl]urea;

(S)-l-(4-(azetidin-l-ylmethyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3- yl)urea;

(S)-l-(4-(azetidin-l-ylmethyl)-3-fluorophenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea;

(S)-l-(3-chloro-4-((dimethylamino)methyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea; (S)-l-(4-(azetidin-l-ylmethyl)-3-chlorophenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea;

(S)-l-(3-chloro-4-((4-methylpiperazin-l-yl)methyl)phenyl)-3-(l-(2-(methylamino)pyrimidm 4-yl)pyrrolidin-3-yl)urea;

(S)-l-(3-chloro-4-((4-ethylpiperazin-l-yl)methyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea;

(S)-l-(3-chloro-4-(morpholinomethyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea;

(S)-l-(4-((dimethylamino)methyl)-3-(trifluoromethyl)phenyl)-3-(l-(2- (methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea;

(S)-l-(4-(azetidin-l-ylmethyl)-3-(trifluor^

4-yl)pyrrolidin-3-yl)urea;

(S)-l-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)-3-(4-(pyrrolidin-l-ylmethyl)-3 (trifluoromethyl)phenyl)urea;

(S)-l-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)-3-(4-((4-methylpiperazin-l- yl)methyl)-3-(trifluoromethyl)phenyl)urea;

(S)-l-(4-((4-ethylpiperazin-l-yl)methyl)-3-(trifluoromethyl)phenyl)-3-(l-(2- (methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea;

(S)-l-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)-3-(4-(morpholinomethyl)-3 (trifluoromethyl)phenyl)urea;

l-(4-Azetidin-l-ylmethyl-3-trifluoromethyl-phenyl)-3-[(S)-l-(2-methylamino-pyrimidm yl)-pyrrolidin-3-yl]-urea;

(S)-l-(l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl)-3-(3-chloro-4- methylphenyl)urea; and

pharaiaceutically acceptable salts thereof.

Another embodiment includes a compound selected from:

and salts thereof.

Uses, Formulation and Administration

Pharmaceutically acceptable compositions

Another aspect includes a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof. In one embodiment, the

composition further comprises a pharmaceutically acceptable carrier, adjuvant, or vehicle. In another embodiment, the composition further comprises an amount of the compound effective to measurably inhibit CDK8. In certain embodiments, the composition is formulated for administration to a patient in need thereof.

The term "patient" or "individual" as used herein, refers to an animal, such as a mammal, such as a human. In one embodiment, patient or individual refers to a human.

The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" refers to a nontoxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

Compositions comprising a compound of formula I or salt thereof may be administered orally, parenterally, by inhalation spray, topically, transdermally, rectally, nasally, buccally, sublingually, vaginally, intraperitoneal, intrapulmonary, intradermal, epidural or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

In one embodiment, the composition comprising a compound of formula I or salt thereof is formulated as a solid dosage form for oral administration. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In certain embodiments, the solid oral dosage form comprising a compound of formula (I) or a salt thereof further comprises one or more of (i) an inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and (ii) filler or extender such as starches, lactose, sucrose, glucose, mannitol, or silicic acid, (iii) binders such as

carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose or acacia, (iv) humectants such as glycerol, (v) disintegrating agent such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates or sodium carbonate, (vi) solution retarding agents such as paraffin, (vii) absorption accelerators such as quaternary ammonium salts, (viii) a wetting agent such as cetyl alcohol or glycerol monostearate, (ix) absorbent such as kaolin or bentonite clay, and (x) lubricant such as talc, calcium stearate, magnesium stearate, polyethylene glycols or sodium lauryl sulfate. In certain embodiments, the solid oral dosage form is formulated as capsules, tablets or pills. In certain embodiments, the solid oral dosage form further comprises buffering agents. In certain embodiments, such compositions for solid oral dosage forms may be formulated as fillers in soft and hard-filled gelatin capsules comprising one or more excipients such as lactose or milk sugar, polyethylene glycols and the like. In certain embodiments, tablets, dragees, capsules, pills and granules of the compositions comprising a compound of formula I or salt thereof optionally comprise coatings or shells such as enteric coatings. They may optionally comprise opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.

Examples of embedding compositions include polymeric substances and waxes, which may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

In another embodiment, a composition comprises micro-encapsulated compound of formula (I) or salt thereof, and optionally, further comprises one or more excipients.

In another embodiment, compositions comprise liquid dosage formulations comprising a compound of formula I or salt thereof for oral administration, and optionally further comprise one or more of pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In certain embodiments, the liquid dosage form optionally, further comprise one or more of an inert diluent such as water or other solvent, a solubilizing agent, and an emulsifier such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols or fatty acid esters of sorbitan, and mixtures thereof. In certain embodiments, liquid oral

compositions optionally further comprise one or more adjuvant, such as a wetting agent, a suspending agent, a sweetening agent, a flavoring agent and a perfuming agent.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of a compound of formula (I), it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled.

Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

In certain embodiments, the composition for rectal or vaginal administration are formulated as suppositories which can be prepared by mixing a compound of formula (I) or a salt thereof with suitable non-irritating excipients or carriers such as cocoa butter,

polyethylene glycol or a suppository wax, for example those which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the compound of formula (I).

Example dosage forms for topical or transdermal administration of a compound of formula (I) include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The compound of formula (I) or a salt thereof is admixed under sterile conditions with a pharmaceutically acceptable carrier, and optionally preservatives or buffers. Additional formulation examples include an ophthalmic formulation, ear drops, eye drops,, transdermal patches. Transdermal dosage forms can be made by dissolving or dispensing the compound of formula (I) or a salt thereof in medium, for example ethanol or

dimethylsulfoxide. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel. Nasal aerosol or inhalation formulations of a compound of formula (I) or a salt thereofmay be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promotors to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

In certain embodiments, pharmaceutical compositions may be administered with or without food. In certain embodiments, pharmaceutically acceptable compositions are administered without food. In certain embodiments, pharmaceutically acceptable compositions of this invention are administered with food.

Specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including age, body weight, general health, sex, diet, time of

administration, rate of excretion, drug combination, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of a provided compound of formula I or salt thereof in the composition will also depend upon the particular compound in the composition.

In one embodiment, the therapeutically effective amount of the compound of the invention administered parenterally per dose will be in the range of about 0.01-100 mg/kg, alternatively about 0.1 to 20 mg/kg of patient body weight per day, with the typical initial range of compound used being 0.3 to 15 mg/kg/day. In another embodiment, oral unit dosage forms, such as tablets and capsules, contain from about 5 to about 100 mg of the compound of the invention.

An example tablet oral dosage form comprises about 2 mg, 5 mg, 25mg, 50mg, lOOmg, 250mg or 500mg of a compound of formula (I) or salt thereof, and further comprises about 95-30 mg anhydrous lactose, about 5-40 mg sodium croscarmellose, about 5-30mg polyvinylpyrrolidone (PVP) K30 and about 1-10 mg magnesium stearate. The process of formulating the tablet comprises mixing the powdered ingredients together and further mixing with a solution of the PVP. The resulting composition can be dried, granulated, mixed with the magnesium stearate and compressed to tablet form using conventional equipment. An example of an aerosol formulation can be prepared by dissolving about 2-500 mg of a compound of formula I or salt thereof, in a suitable buffer solution, e.g. a phosphate buffer, and adding a tonicifier, e.g. a salt such sodium chloride, if desired. The solution may be filtered, e.g. using a 0.2 micron filter, to remove impurities and contaminants.

Uses of Compounds and Pharmaceutically Acceptable Compositions Another aspect includes the use of a compound of formula (I) or a salt thereof for the inhibition of CDK8, thereby treating diseases, such as cancer.

Another aspect includes a method of treating or preventing a disease responsive to the inhibition of CDK8 activity in a patient. The method includes administering a therapeutically effective amount of a compound of formula (I) or a salt thereof to a patient in need thereof.

Another aspect includes the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in therapy. Another aspect includes the use of a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, in therapy.

Another aspect includes the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in treating a disease associated with CDK8 activity.

Another aspect includes the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease associated with CDK8 activity.

Another aspect includes the compound of formula (I), or a pharmaceutically acceptable salt thereof for use as therapeutic active substances.

Another aspect includes the compound of formula (I), or a pharmaceutically acceptable salt thereof for use as therapeutic active substances for the treatment of a hyperproliferative disorder.

In certain embodiments, the disease or condition is a hyperproliferative disease, cancer, stroke, diabetes, hepatomegaly, cardiovascular disease, multiple sclerosis,

Alzheimer's disease, cystic fibrosis, viral disease, autoimmune diseases, atherosclerosis, restenosis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, asthma, allergic disorders, inflammation, neurological disorders, a hormone-related disease, conditions associated with organ transplantation, immunodeficiency disorders, destructive bone disorders, proliferative disorders, infectious diseases, conditions associated with cell death, thrombin-induced platelet aggregation, liver disease, pathologic immune conditions involving T cell activation, CNS disorders or a myeloproliferative disorder.

In certain embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

Another aspect includes a method for treating, ameliorating or preventing cancer, drug-resistant cancer or another proliferative disorder by administration of an effective amount of a compound of formula (I) or salt thereof to a mammal, for example a human, in need of such treatment. In certain embodiments, the disease to be treated is cancer or drug resistant cancer.

Examples of cancers that may be treated using the compounds and methods described herein include, but are not limited to, adrenal cancer, acinic cell carcinoma, acoustic neuroma, acral lentigious melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressive NK- cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, androgen dependent cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear- cell sarcoma of the kidney, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine gland neoplasm, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastric cancer,

hemangioblastoma, head and neck cancer, hemangiopericytoma, hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, leukemia, leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myelogeous leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non-small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor, mucinous tumor, multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, neurinoma, neuroblastoma,

neurofibroma, neuroma, nodular melanoma, ocular cancer, oligoastrocytoma,

oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid cancer,

paraganglioma, pinealoblastoma, pineocytoma, pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, preimary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma periotonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease, small intestine cancer, stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroid cancer, transitional cell carcinoma, throat cancer, urachal cancer, urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macro globulinemia,

Warthin's tumor and Wilms' tumor.

Another embodiment includes a method for the treatment of benign proliferative disorders. Examples of benign proliferative disorders include, but are not limited to, benign soft tissue tumors, bone tumors, brain and spinal tumors, eyelid and orbital tumors, granuloma, lipoma, meningioma, multiple endocrine neoplasia, nasal polyps, pituitary tumors, prolactinoma, pseudotumor cerebri, seborrheic keratoses, stomach polyps, thyroid nodules, cystic neoplasms of the pancreas, hemangiomas, vocal cord nodules, polyps, and cysts, Castleman disease, chronic pilonidal disease, dermatofibroma, pilar cyst, pyogenic granuloma and juvenile polyposis syndrome.

Another embodiment includes the use of a compound of formula I or salt thereof for the production of pharmaceutical compositions which are employed for the treatment and/or prophylaxis and/or amelioration of the diseases, disorders, illnesses and/or conditions as mentioned herein.

Another embodiment includes the use of a compound of formula I or salt thereof for the production of pharmaceutical compositions which are employed for the treatment and/or prophylaxis of diseases and/or disorders responsive or sensitive to the inhibition of CDK8, particularly those diseases mentioned above, such as e.g. cancer.

Compounds of formula (I) or salts thereof may be administered using any amount and any route of administration effective for treating or lessening the severity of the disorder. The exact amount required will vary from patient to patient, depending on the species, age, and general condition of the patient, for example the severity of the disorder, the particular compound, its mode of administration, and the like. The total daily usage of a compound of formula (I) by a given patient will be decided by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts.

Another embodiment includes a method of inhibiting CDK8 activity in a biological sample comprising contacting said biological sample with a compound of formula I or salt thereof.

The term "biological sample", as used herein, includes, without limitation, a cell, cell cultures or extracts thereof; biopsied material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

Co- Administration of Compounds and Other Agents

The compounds of formula (I) or salts therof may be employed alone or in combination with other agents for treatment. For example, the second agent of the pharmaceutical combination formulation or dosing regimen may have complementary activities to the compound of formula (I) such that they do not adversely affect each other. The compounds may be administered together in a unitary pharmaceutical composition or separately. In one embodiment a compound or a pharmaceutically acceptable salt can be coadministered with a cytotoxic agent to treat proliferative diseases and cancer.

The term "co-administering" refers to either simultaneous administration, or any manner of separate sequential administration, of a compound of formula (I) or a salt thereof, and a further active pharmaceutical ingredient or ingredients, including cytotoxic agents and radiation treatment. If the administration is not simultaneous, the compounds are administered in a close time proximity to each other. Furthermore, it does not matter if the compounds are administered in the same dosage form, e.g. one compound may be administered topically and another compound may be administered orally.

Typically, any agent that has activity against a disease or condition being treated may be co-administered. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V.T. Devita and S. Hellman (editors), 6^th edition (February 15, 2001), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the disease involved.

In one embodiment, the treatment method includes the co-administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one cytotoxic agent. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At²¹¹, 1¹³¹, 1¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸,

153 212 32 212

Sm , Bi , P , Pb and radioactive isotopes of Lu); chemo therapeutic agents; growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.

Exemplary cytotoxic agents can be selected from anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormonal analogues, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, nhibitors of LDH-A; inhibitors of fatty acid biosynthesis; cell cycle signaling inhibitors; HDAC inhibitors, proteasome inhibitors; and inhibitors of cancer metabolism.

"Chemotherapeutic agent" includes chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA^®, Genentech/OSI Pharm.), bortezomib (VELCADE^®, Millennium Pharm.), disulfiram , epigallocatechin gallate , salinosporamide A, carfilzomib, 17-AAG(geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX^®, AstraZeneca), sunitib (SUTENT^®, Pfizer/Sugen), letrozole (FEMARA^®, Novartis), imatinib mesylate (GLEEVEC^®., Novartis), finasunate (VATALANIB^®, Novartis), oxaliplatin (ELOXATIN^®, Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin (Sirolimus, RAPAMUNE^®, Wyeth), Lapatinib (TYKERB^®,

GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), sorafenib (NEXAVAR^®, Bayer Labs), gefitinib (IRESSA^®, AstraZeneca), AG1478, alkylating agents such as thiotepa and CYTOXAN^® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;

ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine;

acetogenins (especially bullatacin and bullatacinone); a camptothecin (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenocorticosteroids (including prednisone and prednisolone); cyproterone acetate; 5a- reductases including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat dolastatin; aldesleukin, talc duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,

novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γΐΐ and calicheamicin coll (Angew Chem. Intl. Ed. Engl. 1994 33: 183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as

neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, ADRIAMYCIN (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin,

methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate;

hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK^® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran;

spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;

dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE^® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE^® (docetaxel, doxetaxel; Sanofi-Aventis); chloranmbucil; GEMZAR^® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP- 16); ifosfamide; mitoxantrone;

vincristine; NAVELBINE^® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA^®); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylorni thine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Chemotherapeutic agent also includes (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX ;

tamoxifen citrate), raloxifene, droloxifene, iodoxyfene , 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON^® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE^® (megestrol acetate), AROMASIN^® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR^® (vorozole), FEMARA^® (letrozole; Novartis), and AREVIIDEX^® (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin,

fluoxymesterone, all transretionic acid, fenretinide, as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME^®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN^®, LEUVECTIN^®, and VAXID^®; PROLEUKIN^®, rIL-2; a topoisomerase 1 inhibitor such as LURTOTECAN^®; ABARELIX^® rmRH; and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the above.

Chemotherapeutic agent also includes antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idee), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin

(MYLOTARG®, Wyeth). Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab,

rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and the anti- interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories) which is a recombinant exclusively human- sequence, full-length IgGi λ antibody genetically modified to recognize interleukin-12 p40 protein.

Chemotherapeutic agent also includes "EGFR inhibitors," which refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, and is alternatively referred to as an "EGFR antagonist." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US Patent No. 4,943, 533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab;

ERBUTIX^®) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targeted antibody (Imclone); antibodies that bind type II mutant EGFR (US Patent No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in US Patent No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF or Panitumumab (see WO98/50433, Abgenix/Amgen); EMD 55900

(Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF-alpha for EGFR binding (EMD/Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known as El. l, E2.4, E2.5, E6.2, E6.4, E2.l l, E6. 3 and E7.6. 3 and described in US 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al, J. Biol. Chem. 279(29):30375-30384 (2004)). The anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g.,

EP659,439A2, Merck Patent GmbH). EGFR antagonists include small molecules such as compounds described in US Patent Nos: 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, as well as the following PCT publications: W098/14451, WO98/50038, WO99/09016, and WO99/24037. Particular small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA^® Genentech/OSI Pharmaceuticals); PD 183805 (CI 1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4- morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 4-(3'-Chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(l-methyl-piperidin-4-yl)-pyrimido[5,4- d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(l- phenylethyl)amino]- lH-pyrrolo[2,3-d]pyrimidin-6-yl] -phenol); (R)-6-(4-hydroxyphenyl)-4- [(l-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3- bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4- fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU 5271 ; Pfizer); dual EGFR/HER2 tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016 or N-[3-chloro-4-[(3

fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4- quinazolinamine) .

Chemotherapeutic agents also include "tyrosine kinase inhibitors" including the EGFR-targeted drugs noted in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan- HER inhibitors such as canertinib (CI- 1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf- 1 signaling; non- HER targeted TK inhibitors such as imatinib mesylate (GLEEVEC®, available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib

(PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035,4- (3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H- pyrrolo[2,3-d] pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4- fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner- Lamber); antisense molecules (e.g. those that bind to HER-encoding nucleic acid); quinoxalines (US Patent No. 5,804,396); tryphostins (US Patent No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors such as CI- 1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinib mesylate (GLEEVEC ·); P I 166

(Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-lCl l (Imclone), rapamycin (sirolimus, RAPAMUNE®); or as described in any of the following patent publications: US Patent No. 5,804,396; WO 1999/09016 (American Cyanamid); WO

1998/43960 (American Cyanamid); WO 1997/38983 (Warner Lambert); WO 1999/06378 (Warner Lambert); WO 1999/06396 (Warner Lambert); WO 1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO 1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).

Chemotherapeutic agents also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.

Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone- 17-butyrate, hydrocortisone- 17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate,

prednicarbate, clobetasone- 17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene acetate; immune selective anti-inflammatory peptides (ImSAIDs) such as phenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG) (IMULAN BioTherapeutics, LLC); anti-rheumatic drugs such as azathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine,

leflunomideminocycline, sulfasalazine, tumor necrosis factor alpha (TNFa) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi), Interleukin 1 (IL-1) blockers such as anakinra (Kineret), T cell costimulation blockers such as abatacept (Orencia), Interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®); Interleukin 13 (IL- 13) blockers such as lebrikizumab;

Interferon alpha (IFN) blockers such as Rontalizumab; Beta 7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as Anti-Mi prime; Secreted homotrimeric LTa3 and membrane bound heterotrimer LTal/p2 blockers such as Anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g., At²¹¹, 1¹³¹, 1¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); miscellaneous investigational agents such as thioplatin, PS-341, phenylbutyrate, ET- 18- OCH₃, or farnesyl transferase inhibitors (L-739749, L-744832);

polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and 9- aminocamptothecin); podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate

(FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate

(ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines such as

THERATOPE® vaccine; perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents also include non-steroidal anti-inflammatory drugswith analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib, and valdecoxib. NSAIDs can be indicated for the symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathies, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea, metastatic bone pain, headache and migraine, postoperative pain, mild-to- moderate pain due to inflammation and tissue injury, pyrexia, ileus, and renal colic.

Chemotherapeutic agents also include treatments for Alzheimer's Disease such as donepezil hydrochloride and rivastigmine; treatments for Parkinson's Disease such as L- DOPA/carbidopa, entacapone, ropinrole, pramipexole, bromocriptine, pergolide,

trihexephendyl, and amantadine; agents for treating multiple sclerosis (MS) such as beta interferon (e.g., Avonex^® and Rebif^®), glatiramer acetate, and mitoxantrone; treatments for asthma such as albuterol and montelukast sodium; agents for treating schizophrenia such as zyprexa, risperdal, seroquel, and haloperidol; anti-inflammatory agents such as

corticosteroids, TNF blockers, IL-1 RA, azathioprine, cyclophosphamide, and sulfasalazine; immunomodulatory and immunosuppressive agents such as cyclosporin, tacrolimus, rapamycin, mycophenolate mofetil, interferons, corticosteroids, cyclophophamide, azathioprine, and sulfasalazine; neurotrophic factors such as acetylcholinesterase inhibitors, MAO inhibitors, interferons, anti-convulsants, ion channel blockers, riluzole, and antiparkinsonian agents; agents for treating cardiovascular disease such as beta-blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers, and statins; agents for treating liver disease such as corticosteroids, cholestyramine, interferons, and anti-viral agents; agents for treating blood disorders such as corticosteroids, anti-leukemic agents, and growth factors; and agents for treating immunodeficiency disorders such as gamma globulin.

Additionally, chemotherapeutic agents include pharmaceutically acceptable salts, acids or derivatives of any of chemotherapeutic agents, described herein, as well as combinations of two or more of them.

The amount of both the compound of formula (I) or salt thereof and additional agent (in those compositions which comprise an additional therapeutic agent as described above) that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. In certain embodiments, compositions of this invention are formulated such that a dosage of between 0.01 - 100 mg/kg body weight/day of an inventive can be administered.

The additional therapeutic agent and the compound of formula (I) may act synergistically. Therefore, the amount of additional therapeutic agent in such compositions may be less than that required in a monotherapy utilizing only that therapeutic agent, or there may be fewer side effects for the patient given that a lower dose is used. In certain embodiments, in such compositions a dosage of between 0.01 - 1,000 g/kg body weight/day of the additional therapeutic agent can be administered.

In particular, provided herein are methods of treating cancer in an individual comprising administering to the individual (a) a compound of formula (I) or a

pharmaceutically acceptable salt thereof and (b) a cytotoxic agent (e.g. , targeted therapy, chemotherapy, and/or radiotherapy).

Provided herein are methods of treating cancer in an individual wherein cancer treatment comprising administering to the individual (a) an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and (b) an effective amount of a cytotoxic agent, wherein the cancer treatment has increased efficacy compared to a treatment (e.g., standard of care treatment) comprising administering an effective amount of cytotoxic agent without (in the absence of) the compound of formula (I) or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the methods, the cytotoxic agent is radiotherapy.

In certain embodiments of any of the methods, the compound of formula (I) or a pharmaceutically acceptable salt thereof is concomitantly administered with the cytotoxic agent (e.g. , targeted therapy, chemotherapy, and/or radiotherapy). In certain embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered prior to and/or concurrently with the cytotoxic agent (e.g. , targeted therapy, chemotherapy, and/or radiotherapy).

EXEMPLIFICATION

As depicted in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures, wherein R¹, R², R³, and R^a and R^b are as defined for formula I and its alternative embodiments. It will be appreciated that, although the general methods depict the synthesis of certain compounds of the present invention, the following general methods, and other methods known to one of ordinary skill in the art, can be applied to all compounds and subclasses and species of each of these compounds, as described herein. Scheme

Scheme 2

Scheme 3

(a) 2-iodoacetonitrile, K₂C0₃, acetonitrile, 90°C. (b) 1 -tert-butoxy-N,N,N',N'- tetramethyl-methanediamine, DMF, 90°C. (c) Aniline hydrochloride, DMF, 1 10°C. (d) R^a- guanidine, K₂C0₃, 1 -Butanol, DMF, 120°C. (e) Fe, AcOH, 40°C. (f) DIEA, CH₂CI₂.

Embodiments are illustrated by the following examples.

Examples

HPLC separation method A:

Instrument: Gilson-281,

Column: Gemini 150x30mm with 5μιη particle size

Mobile phase: ACN25%-55% H₂O(+0.05%NH₄OH)

Rate: 25 ml/min,

Monitored Wavelength: 220nm/254 nm,

Run length: 12min/ 15min

Column temperature: 30°C.

HPLC separation method B:

Instrument: BH

Column: Phenomenex Gemini C18 200*25mm*10um

Mobile phase: MeCN: 25%-55%; H₂0 (+0.05% NH OH)

Rate : 25 mL/min

Monitored Wavelength: 220 nm/254 nm

Run length: 12min HPLC separation method C:

Instrument: Gilson GX281

Column: YMC -Actus Triart CI 8 150*30mm

Mobile phase: MeCN: 37 -67 ; H₂0 (+0.05% ammonia solution)

Rate : 25 ml/min

Monitored Wavelength: 220 nm/254 nm

Run length: 10 min/15 min

Example 1:

l-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl]-3-[3- (trifluorometh l)phenyl] urea

Step la) Tert-butyl N-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3- yl]carbamate

A mixture of 6-chloro-[l,2,4]triazolo[4,3-b]pyridazine (200 mg, 1.2940 mmol), tert-butyl N- [(3S)-pyrrolidin-3-yl]carbamate (265 mg., 1.4234 mmol) and N,N-diisopropylethylamine (0.29 mL, 1.6822 mmol) in ethanol (6 mL, 98.6 mmol) was stirred at 70°C for 6h. The mixture was then cooled down and concentrated. The residue was triturated in water and the solid obtained was collected by filtration and washed with water. This was then dried under vacuum to afford 240 mg of tert-butyl N-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6- yl)pyrrolidin-3-yl]carbamate as a white solid. 1H NMR (400 MHz, DMSO) δ 9.18 (s, 1H), 8.03 (d, J = 10.0 Hz, 1H), 7.26 (d, J = 6.6 Hz, 1H), 7.03 (d, J = 10.1 Hz, 1H), 4.14 (dd, J = 11.7, 6.0 Hz, 1H), 3.75 - 3.43 (m, 4H), 2.14 (dq, J = 13.4, 7.0 Hz, 1H), 1.91 (dt, J = 12.5, 6.5 Hz, 1H), 1.39 (s, 9H).

Step lb) (3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-amine hydrochloride

Tert-butyl N-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl]carbamate (240 mg, 0.7884 mmol) was stirred in a solution of hydrogen chloride (4 mol/L) in 1,4-dioxane (9.8 mL., 39.42 mmol) at room temperature for 30 minutes. The mixture was then concentrated and dried under vacuum to afford 190 mg of (3S)-l-([l,2,4]triazolo[4,3- b]pyridazin-6-yl)pyrrolidin-3-amine hydrochloride as a yellow solid that was used for the next step without purification.

Step lc) l-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl]-3-[3- (trifluoromethyl)phenyl] urea

l-isocyanato-3-(trifluoromethyl)benzene (28 mg, 0.150 mmol) was added to a solution of (3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-amine hydrochloride (30 mg, 0.124 mmol) and N-ethyldiisopropylamine (0.065 mL, 0.372 mmol) in methylene chloride (1 mL). The mixture was stirred at room temperature for lh. The mixture was then concentrated and purified by HPLC (conditions) to afford 21.6 mg of l-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin- 6-yl)pyrrolidin-3-yl]-3-[3-(trifluoromethyl)phenyl]urea as a white solid. LCMS m/z= 392.2 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 9.24 - 9.17 (s, 1H), 8.83 - 8.69 (s, 1H), 8.14 - 8.02 (d, J = 10.0 Hz, 1H), 8.02 - 7.94 (s, 1H), 7.53 - 7.38 (m, 2H), 7.29 - 7.18 (d, J = 7.0 Hz, 1H), 7.15 - 7.05 (d, J = 10.0 Hz, 1H), 6.80 - 6.67 (d, J = 6.6 Hz, 1H), 4.42 - 4.26 (q, J = 5.7 Hz, 1H), 3.82 - 3.68 (dd, J = 10.9, 6.0 Hz, 1H), 3.66 - 3.51 (m, 2H), 3.48 - 3.37 (dd, J = 10.9, 4.1 Hz, 1H), 2.30 - 2.16 (dq, J = 13.6, 7.0 Hz, 1H), 2.05 - 1.90 (dq, J = 12.4, 6.2 Hz, 1H).

Example 2:

l-[4 hloro-3-(trifluoromethyl)phenyl]-3-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6- l)pyrrolidin-3-yl]urea

LCMS m/z= 426.1 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 9.26 - 9.13 (s, 1H), 8.93 - 8.79 (s, 1H), 8.16 - 7.97 (m, 2H), 7.65 - 7.45 (s, 2H), 7.16 - 6.98 (d, J = 10.0 Hz, 1H), 6.87 - 6.75 (d, J = 6.6 Hz, 1H), 4.43 - 4.26 (q, J = 5.5 Hz, 1H), 3.79 - 3.66 (dd, J = 11.0, 6.1 Hz, 1H), 3.66 - 3.50 (q, J = 7.1, 6.2 Hz, 2H), 3.47 - 3.37 (m, 1H), 2.30 - 2.17 (dd, J = 13.2, 6.5 Hz, 1H), 2.08 - 1.90 (dd, J = 12.2, 6.1 Hz, 1H). Example 3: l-(3-chlorophenyl)-3-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6- l)pyrrolidin-3-yl]urea

LCMS m/z = 358.2 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 9.23 - 9.12 (s, 1H), 8.84 - 8.68 (s, 1H), 8.11 - 7.99 (d, J = 10.0 Hz, 1H), 7.71 - 7.62 (d, J = 2.2 Hz, 1H), 7.30 - 7.14 (m, 2H), 7.11 - 7.04 (d, J = 10.1 Hz, 1H), 6.98 - 6.90 (d, J = 7.6 Hz, 1H), 6.91 - 6.83 (d, J = 6.7 Hz, 1H), 4.41 - 4.25 (q, J = 5.6 Hz, 1H), 3.79 - 3.65 (dd, J = 10.9, 6.0 Hz, 1H), 3.65 - 3.50 (q, J = 6.6, 5.9 Hz, 2H), 3.46 - 3.37 (dd, J = 10.9, 4.1 Hz, 1H), 2.29 - 2.16 (dq, J = 13.6, 7.2 Hz, 1H), 2.07 - 1.88 (dq, J = 12.6, 6.2 Hz, 1H).

Example 4:

l-[(3R)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl]-3-[3- (trifluorometh l)phenyl] urea

LCMS m/z = 392.2 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 9.26 - 9.18 (s, 1H), 8.78 - 8.65 (s, 1H), 8.12 - 8.02 (d, J = 10.0 Hz, 1H), 8.03 - 7.93 (s, 1H), 7.53 - 7.38 (m, 2H), 7.31 - 7.19 (d, J = 7.0 Hz, 1H), 7.14 - 7.01 (d, J = 10.0 Hz, 1H), 6.80 - 6.65 (d, J = 6.8 Hz, 1H), 4.45 - 4.27 (q, J = 5.7 Hz, 1H), 3.80 - 3.68 (dd, J = 10.9, 6.1 Hz, 1H), 3.65 - 3.52 (m, 2H), 3.49 - 3.37 (dd, J = 10.6, 4.0 Hz, 1H), 2.30 - 2.16 (dd, J = 12.9, 6.5 Hz, 1H), 2.05 - 1.92 (dd, J = 12.6, 6.4 Hz, 1H). Example 5:

l-[4-chloro -(trifluoromethyl)phenyl] -[(3R)-l-([l,2,4]triazolo[4,3-b]pyridazin-

6- l)pyrrolidin-3-yl]urea

LCMS m/z = 426.1 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 9.25 - 9.11 (s, 1H), 8.94 - 8.82 (s, 2H), 8.12 - 8.00 (m, 3H), 7.61 - 7.49 (s, 3H), 7.15 - 6.99 (d, J = 10.0 Hz, 2H), 6.89 - 6.75 (d, J = 6.8 Hz, 2H), 2.30 - 2.15 (m, 2H), 4.41 - 4.25 (q, J = 5.6 Hz, 2H), 3.78 - 3.68 (m, 2H), 2.07 - 1.91 (m, 2H), 3.64 - 3.51 (q, J = 6.9, 6.1 Hz, 3H), 3.48 - 3.37 (d, J = 4.1 Hz, 1H).

Example 6:

l-(3-chlorophenyl)-3-[(3R)-l-([l,2,4]triazolo[4,3-b] ridazin-6-yl)pyrrolidin-3-yl]urea

LCMS m/z = 358.2 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 9.24 - 9.15 (s, 1H), 8.75 - 8.61 (s, 1H), 8.12 - 8.00 (d, J = 10.0 Hz, 1H), 7.70 - 7.61 (s, 1H), 7.29 - 7.14 (m, 2H), 7.13 - 7.03 (d, J = 10.0 Hz, 1H), 6.97 - 6.89 (m, 1H), 6.84 - 6.73 (d, J = 6.6 Hz, 1H), 4.41 - 4.23 (q, J = 5.5 Hz, 1H), 3.79 - 3.65 (dd, J = 10.8, 6.0 Hz, 1H), 3.65 - 3.50 (q, J = 6.4, 5.5 Hz, 2H), 3.45 - 3.36 (dd, J = 10.4, 3.8 Hz, 1H), 2.30 - 2.14 (dq, J = 13.7, 7.2 Hz, 1H), 2.03 - 1.89 (dt, J = 12.4, 6.3 Hz, 1H).

Example 7:

(S)-l-(l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl)-3-(4-((4-ethylpiperazin-l- yl)methyl)-3-(trifluoromethyl)phenyl)urea

LCMS m/z = 518.3 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 9.24 - 9.12 (m, 1H), 8.72 (s, 1H), 8.05 (dd, J = 10.0, 0.9 Hz, 1H), 7.92 (d, J = 2.2 Hz, 1H), 7.60 - 7.52 (m, 1H), 7.51 - 7.43 (m, 1H), 7.08 (d, J = 10.0 Hz, 1H), 6.72 (d, J = 6.6 Hz, 1H), 4.34 (d, J = 5.6 Hz, 1H), 3.73 (dd, J = 11.0, 6.0 Hz, 1H), 3.58 (d, J = 7.0 Hz, 2H), 3.49 (s, 2H), 3.41 (dd, J = 11.0, 4.2 Hz, 1H), 2.43 - 2.16 (m, 10H), 1.98 (t, J = 6.1 Hz, 1H), 0.97 (t, J = 7.1 Hz, 3H).

Example 8:

l-[(3S)-l-(2-aminopyrimidi -4-yl)pyrrolidin-3-yl] -(3-chloro-4-methyl-phenyl)urea

Step 8a) tert-butyl N-[(3S)-l-(2-aminopyrimidin-4-yl)pyrrolidin-3-yl]carbamate

A mixture of tert-butyl N-[(3S)-pyrrolidin-3-yl]carbamate (200 mg, 1.074 mmol), 4- chloropyrimidin-2-amine (167 mg, 1.29 mmol) and N,N'-diisopropylethylamine (0.28 mL, 1.61 mmol) in isopropanol (3 mL) was stirred at 90°C. Then the mixture was cooled to room temperature and water was added. The precipitated solid was collected by filtration, washed with water and dried under vacuum to afford 300 mg of tert-butyl N-[(3S)- l-(2- aminopyrimidin-4-yl)pyrrolidin-3-yl]carbamate as a white solid. This product was used for next step without purification.

Step 8b) 4-[(3S)-3-aminopyrrolidin-l-yl]pyrimidin-2-amine Tert-butyl N-[(3S)- l-(2-aminopyrimidin-4-yl)pyrrolidin-3-yl]carbamate (300 mg, 1.074 mmol) was stirred in hydrogen chloride (4 mol/1) in 1,4-dioxane (2.7 mL, 10.74 mmol) at room temperature for lh. The reaction was then concentrated and dried under vacuum to afford 170 mg of 4-[(3S)-3-aminopyrrolidin- l-yl]pyrimidin-2-amine as a white solid. This product was used for next step without purification.

Steb 8c) l-[(3S)-l-(2-aminopyrimidin-4-yl)pyrrolidin-3-yl]-3-(3-chloro-4-methyl- phenyl)urea

2-chloro-4-isocyanato-l -methyl-benzene (56 mg, 0.278 mmol) was added to a solution of 4- [(3S)-3-aminopyrrolidin-l-yl]pyrimidin-2-amine hydrochloride (50 mg, 0.232 mmol) and N- ethyldiisopropylamine (0.204 mL, 1.159 mmol) in methylene chloride (3 mL). The mixture was stirred at room temperature for lh. The mixture was then concentrated and purified by HPLC (conditions) to afford 55.4 mg of l-[(3S)- l-(2-aminopyrimidin-4-yl)pyrrolidin-3-yl]-3- (3-chloro-4-methyl-phenyl)urea as a white solid. LCMS mJz = 347.2 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 8.83 (s, 1H), 8.12 - 8.00 (m, 1H), 7.73 (d, J = 5.8 Hz, 1H), 7.54 (d, J = 1.4 Hz, 2H), 6.72 (d, J = 6.6 Hz, 1H), 5.90 (s, 2H), 5.74 (d, J = 5.8 Hz, 1H), 4.26 (s, 1H), 3.58 (t, J = 8.1 Hz, 1H), 3.42 (s, 2H), 2.15 (dt, J = 13.8, 6.9 Hz, 1H), 1.91 (dd, J = 11.2, 6.0 Hz, 1H).

Example 9:

l-[(S)-l-(2-Amino-pyrimidin-4-yl)-pyrrolidin-3-yl]-3-(4-chloro-3-trifluoromethyl- -urea

LCMS m/z = 401.1 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 8.83 (s, 1H), 8.12 - 8.00 (m, 1H), 7.73 (d, J = 5.8 Hz, 1H), 7.54 (d, J = 1.4 Hz, 2H), 6.72 (d, J = 6.6 Hz, 1H), 5.90 (s, 2H), 5.74 (d, J = 5.8 Hz, 1H), 4.26 (s, 1H), 3.58 (t, J = 8.1 Hz, 1H), 3.42 (s, 2H), 2.15 (dt, J = 13.8, 6.9 Hz, 1H), 1.91 (dd, J = 11.2, 6.0 Hz, 1H). Example 10:

l-[4-chloro-3-(trifluoromethyl)phenyl]-3-[(3S)-l-[2-(methylamino)pyrimidin-4- yl]pyrrolidin-3-yl]urea

LCMS m/z = 415.2 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 8.83 (s, IH), 8.06 (s, IH), 7.76 (d, J = 5.8 Hz, IH), 7.54 (d, J = 1.4 Hz, 2H), 6.73 (d, J = 6.7 Hz, IH), 6.32 (d, J = 5.0 Hz, IH), 5.73 (d, J = 5.9 Hz, IH), 4.27 (s, IH), 3.52 (d, J = 64.8 Hz, 4H), 2.73 (d, J = 4.8 Hz, 3H), 2.16 (dd, J = 12.5, 6.5 Hz, IH), 1.92 (t, J = 8.1 Hz, IH).

Example 11:

l-(3-chloro-4-methyl-phenyl)-3-[(3S)-l-[2-(methylamino)pyrimidin-4-yl]pyrrolidin-3- yl]urea

LCMS m/z = 361.2 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 8.41 (s, IH), 7.76 (d, J = 5.8 Hz, IH), 7.63 (d, J = 2.1 Hz, IH), 7.22 - 7.12 (m, IH), 7.08 (dd, J = 8.3, 2.2 Hz, IH), 6.54 (d, J = 6.8 Hz, IH), 6.32 (q, J = 4.8 Hz, IH), 5.73 (d, J = 5.8 Hz, IH), 4.25 (s, IH), 3.59 (s, IH), 3.43 (s, 3H), 2.73 (d, J = 4.7 Hz, 3H), 2.23 (s, 3H), 2.14 (dt, J = 13.9, 7.7 Hz, IH), 1.89 (s, IH). Example 12:

l-[(3S)-l-[2- (methylamino)pyrimidin-4-yl] pyrrolidin-3-yl] -3- [4- (trifluoromethoxy)phenyl] urea

LCMS m/z = 397.2 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 8.53 (s, 1H), 7.76 (d, J = 5.8 Hz, 1H), 7.54 - 7.39 (m, 2H), 7.22 (d, J = 8.5 Hz, 2H), 6.56 (d, J = 6.8 Hz, 1H), 6.32 (d, J = 5.3 Hz, 1H), 5.73 (d, J = 5.8 Hz, 1H), 4.27 (s, 1H), 3.59 (s, 1H), 3.44 (s, 2H), 2.73 (d, J = 4.7 Hz, 3H), 2.16 (dd, J = 13.0, 6.8 Hz, IH), 1.89 (s, IH).

Example 13:

l-[4-(difluoromethoxy)phenyl]-3-[(3S)-l-[2-(methylamino)pyrimidin-4-yl]pyrrolidin-3- yl]urea

LCMS m/z = 379.2 [M+l]⁺. 1H NMR (400 MHz, DMSO) δ 8.39 (s, IH), 7.76 (d, J = 5.8 Hz, IH), 7.47 - 7.33 (m, 2H), 7.11 - 6.99 (m, 2H), 6.50 (d, J = 6.8 Hz, IH), 6.32 (d, J = 5.3 Hz, IH), 5.73 (d, J = 5.8 Hz, IH), 4.26 (s, IH), 3.57 (s, 4H), 2.73 (d, J = 4.7 Hz, 3H), 2.23 - 2.05 (m, IH), 1.89 (s, IH).

Example 14:

(S)-l-(4-(azetidin-l-ylmethyl)phenyl) -(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3- yl)urea

-(4-nitrobenzyl)azetidine

To a solution of azetidine hydrochloride (1.4 g, 15 mmol) and DIPEA (1.9 g, 15 mmol) in CH₃CN (30 mL) was added l-(bromomethyl)-4-nitrobenzene (1.07 g, 5 mmol) dropwise over 30 min. The reaction mixture was stirred at room temperature for 3 hours under an atmosphere of N₂, and then diluted with DCM (80 mL). The organic layer was washed with water (3 x 80 mL) and concentrated to give the crude title compound, which was carried on without further purification (0.77 g, yield: 81%). -(azetidin-l-ylmethyl)aniline

To a solution of l-(4-nitrobenzyl)azetidine (0.77 g, 4 mmol) in AcOH (10 mL) was added Fe (2.24 g, 40 mmol), and the reaction mixture was stirred at room temperature for 3 h under an atmosphere of N₂. When the starting material was consumed, the reaction mixture was concentrated and basified to a pH of approximately 9, and then extracted with DCM (3x30 mL). The combined organics were concentrated to give the title compound , which was carried on without further purification (453 mg, yield: 71 ). -tert-butyl (l-(2-chloropyrimidin-4-yl)pyrrolidin-3-yl)carbamate

To a solution of (S)-tert-butyl (l-(2-chloropyrimidin-4-yl)pyrrolidin-3-yl)carbamate (14 g, 94.6 mmol) in propan-2-ol (50 mL) was added DIPEA (14.64 g, 113.51 mmol), and the mixture was cooled to 0 °C before (S)-tert-butyl pyrrolidin-3-ylcarbamate (17.6 g, 94.6 mmol) was added to the solution in portions. The reaction solution was stirred at 25 °C for 30 minutes. The mixture was then concentrated, and the residue was purified on a silica gel column (eluting with 10% ethyl acetate in petroleum ether) to give (S)-tert-butyl (l-(2- chloropyrimidin-4-yl)pyrrolidin-3-yl) carbamate (22.5 g, yield 80%) as a solid & (S)-tert- butyl (l-(4-chloropyrimidin-2-yl)pyrrolidin-3-yl)carbamate (3.0 g, yield 10%).

(S)-tert-butyl (l-(2-chloropyrimidin-4-yl)pyrrolidin-3-yl) carbamate

1H NMR (CDC1₃, 400 MHz): δ 7.97 (d, J=6.0, 1H), 6.15 (d, J=6.0, 1H),4.79 (s, 1H), 4.31 (s, 1H), 3.22-3.82 (m, 4H), 2.25 (s, 1H), 1.91-2.03 (m, 1H), 1.42 (s, 9H);

MS (ESI): m/z 298.9 [M+l]⁺.

(S)-tert-butyl (l-(4-chloropyrimidin-2-yl)pyrrolidin-3-yl)carbamate

1H NMR (CDCI₃, 400 MHz): δ 8.09 (d, J=5.2, 1H), 6.45 (d, J=5.2, 1H),4.60 (s, 1H), 4.26 (s, 1H), 3.74-3.79 (m, 1H), 3.53-3.63 (m, 2H), 3.36-3.40 (m, 1H), 2.15-2.23 (m, 1H), 1.85- 1.93 (m, 1H), 1.38 (s, 9H);

MS (ESI): m/z 298.9 [M+l]⁺. -tert-butyl (l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)carbamate

To a solution of (S)-tert-butyl (l-(2-chloropyrimidin-4-yl) pyrrolidin-3-yl)carbamate (22 g, 74 mmol) in methanamine/THF (2 M, 100 ml) was added CuS0₄ (1.18 g, 7.4 mmol). The reaction solution was stirred at 80 C in an autoclave for 16 hours. The mixture was concentrated and the residue was dissolved in ethyl acetate (600 ml), which was washed with NH₄OH solution (3 x 20ml). The organic layer was dried over sodium sulfate and concentrated. The residue was purified on a silica gel column (eluting with 45% ethyl acetate in petroleum ether) to give (S)-tert-butyl (l-(2-(methylamino) pyrimidin-4-yl) pyrrolidin-3- yl)carbamate (21 g, yield 97%).

1H NMR (DMSO-d₆, 400 MHz): δ 7.73 (d, J=5.6, 1H), 7.18 (d, J=5.6, 1H), 6.33 (d, J=4.4, 1H), 5.68 (d, J=5.6, 1H), 4.02-4.05 (m, 1H), 3.54 (m, 4H), 2.72 (d, J=4.8, 3H), 2.07-2.08 (m, 1H), 1.78-1.84 (m, 1H), 1.39 (s, 9H);

MS (ESI): m/z 294.1 [M+l]⁺. -4-(3-aminopyrrolidin-l-yl)-N-methylpyrimidin-2-amine

(S)-tert-butyl (l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)carbamate (21 g, 71.5 mmol) was stirred in 4N HCl/dioxane (50 mL) at room temperature for 1 hour. The reaction mixture was concentrated to give (S)-4-(3-aminopyrrolidin-l-yl)-N-methylpyrimidin-2-amine (18, yield over 100%) as the hydrochloride salt.

1H NMR (DMSO-d₆, 400 MHz): δ 7.74 (d, J=5.6, 1H), 7.34 (d, J=4.8, 1H), 5.68 (d, J=5.2, 1H), 3.63-3.67 (m, 1H), 3.34-3.57 (m, 4H), 2.72 (d, J=4.8, 3H), 2.07-2.09 (m, 1H), 1.77-1.85 (m, 1H).

Step 14 f) (S)-l-(4-(azetidin-l-ylmethyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea

To a solution of 4-(azetidin-l-ylmethyl) aniline (162 mg, 1 mmol) in DMF (4 mL) was added TEA (303 mg, 3 mmol) and CDI (194 mg, 1.3 mmol). The reaction was stirred at room temperature for 30 minutes and then (S)-4-(3-aminopyrrolidin-l-yl)-N-methylpyrimidin-2- amine (332 mg, 1.0 mmol) was added. The mixture was stirred for an additional 2 hours. After the starting material was consumed, the crude product was purified by preparative HPLC under conditions A to give (S)-l-(4-(azetidin-l-ylmethyl)phenyl)-3-(l-(2- (methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea (99.2 mg, 25.9%).

1H NMR (400 MHz, DMSO-d6) δ 8.28 (s, 1 H), 7.76 (d, J = 5.6 Hz, 1 H), 7.29 (d, J = 8.4 Hz, 2 H), 7.09 (d, J = 8.4 Hz, 2 H), 6.49 (d, J = 6.8 Hz, 1 H), 6.35 (d, J = 4.8 Hz, 1 H), 5.74 (d, J = 6.0 Hz, 1 H), 4.24 (brs, 1 H), 3.68-3.21 (m, 6 H), 3.05 (t, J = 6.8 Hz, 4 H), 2.81-2.65 (m, 3 H), 2.12 (m,l H), 1.97-1.82 (m, 3 H). LCMS (ESI): m/z 382.2 [M+H⁺]. Example 15:

(S)-l-(4-(azetidin-l-ylmethyl)-3-fluorophenyl) -(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea

-ylmethyl)-3-fluorophenyl)-2,2,2-trifluoroacetamide

To a solution of azetidine hydrochloride (1.4 g, 15 mmol) and DIPEA (1.9 g, 15 mmol) in CH₃CN (30 mL) was added N-(4-(bromomethyl)-3-fluorophenyl)-2,2,2-trifluoroacetamide (1.5 g, 5 mmol) dropwise over 30 minutes.The reaction mixture was stirred at room

temperature for 3 h under an atmosphere of N₂. After the starting material was consumed, the reaction mixture was diluted with DCM (80 mL), and the organic layer was washed with water (3 x 80 mL), then concentrated to give the title compound which was used without further purification (1.1 g, yield: 81 ). -(azetidin-l-ylmethyl)-3-fluoroaniline

To a solution of N-(4-(azetidin-l-ylmethyl)-3-fluorophenyl)-2,2,2-trifluoroacetamide (1.1 g, 3.9 mmol) in CH₃OH (30 ml) was added a solution of K₂C0₃ (1.62g, 11.7 mmol) in water (10 ml), and the reaction mixture was stirred at 75 C for 3 hours under an atmosphere of N₂. After the starting material was consumed, the reaction mixture was diluted with DCM (80 ml), and the organic layer was washed with water (3 x 80 ml), then concentrated to give the title compound which was used without further purification (645 mg, yield: 91 %). Step 15c) (S)-l-(4-(azetidin-l-ylmethyl)-3-fluorophenyl)-3-(l-(2- (methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea

To a solution of 4-(azetidin-l-ylmethyl)-3-fluoroaniline (180 mg, 1 mmol) in DMF (4 ml) were added TEA (303 mg, 3 mmol) and CDI (194 mg, 1.3 mmol). The reaction was stirred at room temperature for 30 minutes. (S)-4-(3-aminopyrrolidin-l-yl)-N-methylpyrimidin-2- amine (332 mg, 1.0 mmol) was added, and the mixture was stirred for another 2 hours. After the starting material was consumed, the crude product was purified by preparative-HPLC using conditions A to give the title compound (37.1 mg, 9.3%).

1H NMR (400 MHz, DMSO-d₆) δ 8.54 (s, 1 H), 7.81-7.65 (m, 1H), 7.41-6.85 (m, 3 H), 6.65- 6.22 (m, 2 H), 5.74 (d, J = 7.2 Hz, 1 H), 4.26 (brs, 1 H), 3.82-3.01 (m, 9 H), 2.89-2.42 (m, 4 H), 2.32-1.71 (m, 4 H). LCMS (ESI): m/z 400.2 [M+H⁺].

Example 16:

(S)-l-(3-chloro-4-((dimethylamino)methyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea

Step 16a) N-(3-chloro-4-methylphenyl)-2,2,2-trifluoroacetamide

To a solution of 3-chloro-4-methylaniline (14.2 g, lOOmmol) dissolved in DCM (250 ml), was added pyridine (75 ml, 0.80mol) and added dropwise trifluoroacetic anhydride (16.5ml, 112.5mmol) at 0 C .The mixture was stirred at room temperature for 1 hour. The reaction mixture was washed with 10% aqueous HC1, then with water. The organic phase was dried over Na₂S0₄ and concentrated to give the title compound, which was used without further purification (22g ,92.8%). Step 16b) N-(4-(bromomethyl)-3-chlorophenyl)-2,2,2-trifluoroacetamide

To a solution of N-(3-chloro-4-methylphenyl)-2,2,2-trifluoroacetamide (22 g, 92.2 mmol) in CC1₄ (740 ml) was added BPO (6.7g, 27.7 mmol) and NBS (19.7g, 110.6mmol). The mixture was warmed to 100°C and illuminated for 1 h by a high voltage lamp. The reaction mixture was stirred at 100 C for 36 h. After cooling down to 0°C, the reaction mixture was filtered and the filtrate was washed with brine, then concentrated. The crude product was washed with petroleum ether and filtered to give the title compound, which was used without further purification (14 g, 48.3%).

Step 16c) N-(3-chloro-4-((dimethylamino)methyl)phenyl)-2,2,2-trifluoroacetamide

To a solution of dimethylamine hydrochloride (4.6g, 56.4 mmol) in MeCN (150 ml) was added dropwise N-(4-(bromomethyl)-3-chlorophenyl)-2,2,2-trifluoroacetamide (3.0 g, 9.4 mmol) dissolved in MeCN (150 ml) over lhour at 0°C, and stirred at 0°C for 30minutes. Subsequently, 100 mL of saturated NaHC0₃ was added, and the mixture was partitioned between DCM and water. The organic layer was washed with water (15 mL), dried over Na₂S0₄, concentrated and purified by prep-TLC (petroleum ethenEtOAc = 2: 1) to give the title compound(2.2g, 83%). -chloro-4-((dimethylamino)methyl)aniline

To a solution of N-(3-chloro-4-((dimethylamino) methyl) phenyl)-2,2,2-trifluoroacetamide (2.2 g, crude) in MeOH (30 mL) and H₂O(30ml) was added K₂C0₃(3.25g, 23.6mmol) at room temperature . The mixture was then stirred at 80°C for 1 hour. The reaction mixture was concentrated to give the title compound (1.3g, 90.1%).

1H NMR (400 MHz, CDC1₃) 57.48(d,J=8.0Hz,lH) , 56.91 (d,J=2.4Hz,lH) , 6.79 -6.77(m, 1H) , 53.77 (s,2H) , 53.52 (s,2H), 52.47 (d,J=10.8Hz,7H), 52.28 (s,3H). Step 16e) (S)-l-(3-chloro-4-((dimethylamino)methyl)phenyl)-3-(l-(2-

(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea

To a solution of 3-chloro-4-((dimethylamino) methyl) aniline (200 mg, 1.08mmol) in dry DMF (5 ml) was added CDI (224mg, 1.4 mmol), and DIPEA (413.2mg, 3.26mmol)at 25°C. The mixture was stirred at 25°C for 2hours. (S)-4-(3-aminopyrrolidin-l-yl)-N- methylpyrimidin-2-amine (208mg, 1.08 mmol) was then added, and the mixture was stirred at 25°C for 16hours. The mixture was purified by prep-HPLC (method B) to give title compound (22.0 mg, 5.0%).

1H NMR (400 MHz, DMSO- ₆) δ 8.42(s,lH) , 57.74 (d,j=6.0Hz,lH) , 7.62(d,j=2.0Hz,lH) , 7.24 (d,j=8.4Hz,lH), 7.13-7.11 (m, 1H), 6.55(d,j=6.8Hz,lH), 6.33(d,j=4.4Hz,lH),

5.71(d,j=5.6Hz,lH), 4.23 (s,lH), 3.55 (d,j=2.8Hz,2H), 3.34(s,3H), 2.71(d,j=4.8Hz,3H), 2.12(s,8H), 1.87(d,j=9.6Hz, 1H)

LCMS (ESI): m/z 404.0 [M+H⁺].

Example 17:

(S)-l-(4-(azetidin-l-ylmethyl) -chlorophenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea

1H NMR (400 MHz, CDC1₃) 5 8.09 (brs, 1 H), 7.67 (d, J = 6.0 Hz, 1 H), 7.49 (s, 1 H), 7.31- 7.17 (m, 2 H), 6.67 (brs, 1 H), 5.57 (d, J = 6.0 Hz, 1 H), 4.78 (brs, 1 H), 4.50 (brs, 1 H), 3.62 (s, 1 H), 3.62-2.85 (m, 9 H), 2.35-2.11 (m, 4 H), 1.27 (s, 2 H). LCMS (ESI): m/z 416.2

[M+H⁺]. Example 18:

(S)-l-(3-chloro-4-((4-methylpiperazin-l-yl)methyl)phenyl)-3-(l-(2- (methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea

1H NMR (400 MHz, DMSO- ₆) δ 8.48(s,lH) , 57.74 (d,j=5.6Hz,lH) , 7.62(d,j=2.4Hz,lH) , 7.25 (d,j=8.4Hz,lH), 7.13-7.10 (m, 1H), 6.58(d,j=6.8Hz,lH), 5.71(d,j=6.0Hz,lH), 4.23 (s,lH), 3.561 (d,j=3.6Hz,lH), 3.42(s,3H), 2.71(d,j=4.8Hz,3H), 2.50(d,j=2.0Hz,2H) ,2.35- 2.28(m,8H), 2.14 (s,lH), 2.11(s,3H), 1.89-1.84(m,lH)

LCMS (ESI): m/z 459.0 [M+H⁺].

Example 19:

(S)-l-(3-chloro-4-((4-ethylpiperazin-l-yl)methyl)phenyl)-3-(l-(2- (methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea

Step 19a) N-(3-chloro-4-((4-ethylpiperazin-l-yl)methyl)phenyl)-2,2,2-trifluoroacetamide

To a solution of N-(4-(bromomethyl)-3-chlorophenyl)-2,2,2-trifluoroacetamide (2.0g, 6.35 mmol) in MeCN (150 ml) was added dropwise 1-ethylpiperazine (4.3g, 38.1 mmol) dissolved in MeCN (150 ml) over lhr at 0°C.The mixture was stirred at 0°C for 30min . After 100 ml of saturated NaHC0₃ was added, the mixture was partitioned between DCM and water. The organic layer was washed with water (15 ml), dried over Na₂S0₄, concentrated and purified by prep-TLC (PE: EA = 2: 1) to give title compound used for next step without further purification (1.8 g, 81.8%).

Step 19b) 3-chloro-4-((4-ethylpiperazin-l-yl)methyl)aniline

3-chloro-4-((4-ethylpiperazin-l-yl)methyl)aniline was prepared analogously to 3-chloro-4- ((dimethylamino)methyl)aniline substituting 1-ethylpiperazine for dimethylamine

hydrochloride.

1H NMR (400 MHz, CDC1₃) 57.01(d, J=7.6Hz, 1H) , 6.55 (d, J=1.2Hz, 1H) , 6.45(m, 1H), 5.23(s, 2H) , 2.23-2.33 (m, 10 H), 0.93 (m, 3H).

Step 19c) phenyl (3-chloro-4-((4-ethylpiperazin-l-yl)methyl)phenyl)carbamate

To a solution of 3-chloro-4-((4-ethylpiperazin-l-yl)methyl)aniline (300 mg, 1.18mmol) in dry THF (12 ml) was added dropwise phenyl chloroformate (185mg, 1.18 mmol) at 65°C. The mixture was stirred at 65°C for 30 minutes. After cooling to 25°C, the mixture was filtered to give the title compound, which was used without further purification (290mg, 66%).

Step 19d) (S)-l-(3-chloro-4-((4-ethylpiperazin-l-yl)methyl)phenyl)-3-(l-(2- (methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea

To a solution of phenyl (3-chloro-4-((4-ethylpiperazin-l-yl)methyl)phenyl)carbamate (290 mg) in dry DMF (3 ml) was added DIPEA (102.7mg, 0.79mmol) at room temperature. The mixture was stirred at room temperature for 0.5h. (S)-4-(3-aminopyrrolidin-l-yl)-N- methylpyrimidin-2-amine (150 mg, 0.40 mmol) was added, and the mixture was stirred at room temperature for 16h. The mixture was purified by prep-HPLC (method C) to give the title compound (18.0 mg, 3.2%).

1H NMR (400 MHz, DMSO- ₆) δ 8.45(s, 1H), 7.74 (d, J=6.0Hz, 1H), 7.62(d, J=2.0Hz, 1H), 7.25 (d, J=8.8Hz, 1H), 7.12-7.10 (m, 1H), 6.55(d, J=6.8Hz, 1H), 6.33-6.30(m, 1H) , 5.71(d, J=6.4Hz, 1H), 3.57-3.54(m, 2H), 3.41(s, 3H),3.37-3.32(m, 2H), 3.27(s, 1H), 2.7 l(d, J=4.8Hz, 3H), 2.52(d, 1H), 2.42-2.23 (m, 10H), 2.13-2.10 (m, 1H), 1.87 (d, J=7.2Hz, 1H), 0.94 (t, J=7.2Hz, 3H)

LCMS (ESI): m/z 473.0 [M+H⁺]. Example 20:

(S)-l-(3-chloro-4-(morpholinomethyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea

This compound was prepared according the general procedure used to make (S)-l-(3- chloro-4-((4-ethylpiperazin-l-yl)methyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea (Example 19).

1H NMR (400 MHz, DMSO- ₆) δ Ί .65-1.62 (m, 2 H), 7.36 (d, J = 8.4 Hz, 1 H), 7.22 (dd, J = 2.4, 8.4 Hz, 1 H), 6.17 (s, 1 H), 4.46-4.40 (m, 1 H), 3.95-3.82 (m, 2 H), 3.71-3.68 (m, 8 H), 3.48-3.47 (m, 1 H), 2.96 (s, 3 H), 2.61-2.59 (m, 4 H), 2.38-2.32 (m, 1 H), 2.12-2.04 (m, 1 H). LCMS (ESI): m/z 446.0 [M+H⁺].

Example 21:

(S)-l-(4-((dimethylamino)methyl)-3-(trifluoromethyl)phenyl)-3-(l-(2- (methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea

This compound was prepared according the general procedure used to make (S)-l-(4- (azetidin-l-ylmethyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea (Example 14).

1H NMR (400 MHz, CDC1₃) δ 8.22 (br s, 1 H), 7.72-7.51 (m, 4 H), 7.59-7.45 (m, 2 H), 6.83 (br s, 1 H), 5.61-5.57 (m, 1 H), 4.99 (brs, 1 H), 4.53 (brs, 1 H), 3.82-3.25 (m, 6 H), 2.93 (d, J = 4.4 Hz, 3 H), 2.36-2.05 (m, 8 H). LCMS (ESI): m/z 438.2 [M+H⁺]. Example 22:

(S)-l-(4-(azetidin-l-ylmethyl)-3-(trifluoromethyl)phenyl)-3-(l-(2- (methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea

This compound was prepared according the general procedure used to make (S)-l-(4- (azetidin-l-ylmethyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea (Example 14).

1H NMR (400 MHz, CDC1₃) δ 8.22 (brs, 1 H), 7.73-7.61 (m, 2 H), 7.59-7.45 (m, 2 H), 6.82 (brs, 1 H), 5.62-5.51 (m, 1 H), 4.84 (brs, 1 H), 4.51 (brs, 1 H), 3.71 (s, 2 H), 3.72-3.05 (m, 8 H), 2.93 (d, J = 4.8 Hz, 3 H), 2.32-2.05 (m, 4 H). LCMS (ESI): m/z 450.2 [M+H⁺].

Example 23:

(S)-l-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)-3-(4-(pyrrolidin-l-ylmethyl)-3-

(trifluoromethyl)phenyl)urea

This compound was prepared according the general procedure used to make (S)-l-(4- (azetidin-l-ylmethyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea (Example 14).

1H NMR (400 MHz, CDC1₃) δ 8.12 (brs, 1 H), 7.72-7.52 (m, 4 H), 7.59-7.45 (m, 2 H), 6.89 (brs, 1 H), 5.64-5.62 (m, 1 H), 5.39 (brs, 1 H), 4.59 (brs, 1 H), 3.76 (s, 3 H), 3.72-3.41 (m, 3 H), 2.92 (d, J = 4.0 Hz, 3 H), 2.58 (s, 4 H), 2.36-1.85 (m, 6 H). LCMS (ESI): m/z 464.2

[M+H⁺].

Example 24:

(S)-l-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)-3-(4-((4-methylpiperazin-l- thyl)-3-(trifluoromethyl)phenyl)urea

This compound was prepared according the general procedure used to make (S)-l-(3- chloro-4-((4-ethylpiperazin-l-yl)methyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea (Example 19).

1H NMR (400 MHz, DMSO- ₆) δ 8.83 (s, 1 H), 8.16 (s, 2 H), 7.90 (d, J = 2.0 Hz, 1 H), 7.74 (d, J = 6.0 Hz, 1 H), 7.52 (d, J = 8.4 Hz, 1 H), 7.46-7.44 (m, 1 H), 6.79 (d, J = 1.2 Hz, 1 H), 6.48 (brs, 1 H), 5.73 (d, J = 6.0 Hz, 1 H), 4.24 (s, 2 H), 3.58-3.55 (m, 2 H), 3.43 (s, 3 H), 2.71 (d, J = 4.8 Hz, 3 H), 2.48-2.37 (m, 8 H), 2.20 (s, 3 H), 2.15-2.13 (m, 1 H), 1.89-1.86 (m, 1 H).

LCMS (ESI): m/z 493.1 [M+H⁺].

Example 25:

(S)-l-(4-((4-ethylpiperazin-l-yl)methyl)-3-(trifluoromethyl)phenyl)-3-(l-(2- (methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea

This compound was prepared according the general procedure used to make (S)-l-(4- (azetidin-l-ylmethyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea (Example 14).

1H NMR (400 MHz, Methanol-^) ) δ 7.81 (s, 1 H), 7.74 (d, J = 6.0 Hz, 1 H), 7.65 (d, J = 8.8 Hz, 1 H), 7.53 (d, J = 8.0 Hz, 1 H), 5.84 (d, J = 6.4 Hz, 1 H), 4.62-4.51 (m, 2 H), 4.49-4.32 (m, 1 H), 3.61-3.37 (m, 5 H), 2.88 (s, 3 H), 2.65- 1.95 (m, 11 H), 1.12 (t, J = 7.2 Hz, 3 H). LCMS (ESI): m/z 507.2 [M+H⁺]. Example 26:

(S)-l-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)-3-(4-(morpholinomethyl)-3-

(trifluoromethyl)phenyl)urea

This compound was prepared according the general procedure used to make (S)-l-(3- chloro-4-((4-ethylpiperazin-l-yl)methyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea (Example 19).

1H NMR (400 MHz, DMSO- ₆) δ 8.80 (s, 1 H), 7.93 (s, 1 H), 7.77 (d, J = 6.0 Hz, 1 H), 7.58 (d, J = 8.4 Hz, 1 H), 7.49-7.47 (m, 1 H), 6.76 (d, J = 6.4 Hz, 1 H), 5.81 (d, J = 6.0 Hz, 1 H), 4.27 (s, 2 H), 3.65-3.62 (m, 1 H), 3.58-3.56 (m, 5 H), 3.51 (s, 3 H), 2.75 (d, J = 8.8 Hz, 3 H), 2.35 (d, J = 4.4 Hz, 4 H), 2.18-2.17 (m, 1 H), 1.92-1.91 (m, 1 H).

LCMS (ESI): m/z 480.2 [M+H⁺].

Example 27:

l-(4-Azetidin-l-ylmethyl-3-trifluoromethyl-phenyl)-3-[(S)-l-(2-methylamino-pyrimidin- -yl)-pyrrolidin-3-yl]-urea

This compound was prepared according the general procedure used to make (S)-l-(4- (azetidin-l-ylmethyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea (Example 14).

1H NMR (400 MHz, CDC1₃) δ 8.22 (brs, 1 H), 7.73-7.61 (m, 2 H), 7.59-7.45 (m, 2 H), 6.82 (brs, 1 H), 5.62-5.51 (m, 1 H), 4.84 (brs, 1 H), 4.51 (brs, 1 H), 3.71 (s, 2 H), 3.72-3.05 (m, 8 H), 2.93 (d, J = 4.8 Hz, 3 H), 2.32-2.05 (m, 4 H). LCMS (ESI): m/z 450.2 [M+H⁺].

Assessment of Inhibitory Effect of Test Compounds on CDK8 activity Inhibition of CDK8 was evaluated in a competitive ATP-site binding assay.¹ Dimers of full length recombinant human His-tagged CDK8 and CyclinC were pre-mixed with europium-labeled anti-His (Life Technologies, Madison, WI) and added to assay plates containing test or control compounds diluted to a range of concentrations. Tracer 236, an Alexa 647-labeled proprietary kinase inhibitor (Life Technologies), was added and incubated for another 60 minutes. Binding of the tracer to CDK8 was detected by Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET). The europium was excited by light at 340 nm resulting in energy transfer to Alexa 647 with subsequent emission of fluorescence at 665 nm when the two fluorophores were brought into close proximity by the binding interaction. Fluorescence intensities (excitation at 340 nm and emission at both 615 and 665 nm) were read on a ViewLux multimode plate reader (PerkinElmer, Waltham, MA). The TR-FRET signal was calculated by dividing the fluorescence intensity at 665 nm by the fluorescence intensity at 615 nm.

Assays were conducted in 1536-well black microplates in volumes of 4 μϊ_^ per well. Final concentrations of CDK8/Cyclin C dimer, anti-His, and Tracer 236 were 5, 4 and 10 nM, respectively. Assay buffer was 50 mM HEPES pH 7.5, 10 mM MgCl₂, 0.01% Brij35, 1 mM EGTA, 0.1% bovine gamma gobulins, and 2 mM dithiothreitol.

Maximal tracer binding, and consequently maximal TR-FRET signal, was obtained in control wells with DMSO as sample. Displacement of tracer by compound resulted in reduced signal. Minimal signal was obtained in control wells lacking the CDK8/Cyclin C dimer. Percent inhibition of tracer binding in the presence of compound was calculated and plotted as a function of compound concentration. The resulting data were fit with 4- parameter Hill curves and potencies were determined as IC₅₀ values using Genedata Screener software (Genedata, Basel, Switzerland).

References:

1. Lebakken CS, Riddle SM, Singh U, Frazee WJ. Et al. Development and applications of a braod-coverage, TR-FRET-based kinase binding assay platform. J. Biomol Screen.

2. Casamassimi A, Napoli C. Mediator complexes and eukaryotic transcription

regulation: an overview. Biochemie (200&) 89, 1439-1446.

3. Alarcon C, Zaromytidou A-I, Xi Q, Gao S, Yu J, et al. Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-β pathways. Cell (2009) 139, 757-769. 4. Morris EJ, Ji J-Y, Yang F, Di Stefano L, Herr A, et al. E2F1 represses β-catenin

transcription and is antagonized by both pRB and CDK8. Nature (2008) 455, 552- 558.

5. Zho J, Ramos R, and Demma M. CDK8 regulates E2F1 transcriptional activity

through S375 phosphorylation. Oncogene (2012), 1-11.

6. Firestein R, Bass AJ, Kim SY, Dunn IF, Silver SJ, et al. CDK8 is a colorectal cancer

oncogene that regulates β-catenin activity. Nature (2008) 455, 547-551.

7. Firestein R and Hahn WC. Revving the throttle on an oncogene: CDK8 takes the

driver seat. Cancer Res (2009) 69, 7899-7901.

8. Adler AS, McCleland ML, Truong T et al. CDK8 maintains tumor de-differentiation

and embryonic stem cell pluripotency. Cancer Research (2012)

9. Xu W and Ji J-Y. Dysregulation of CDK8 and Cyclin C in tumorigenesis. J Genetics

Genomics (2011) 38, 439-452.

10. Firestein R, Shima K, Nosho K, Irahara N, et al. CDK8 expression in 470 colorectal

cancers in relation to β-catenin activation, other molecular alterations and patient survival. International Journal of Cancer (2010) 126, 2863-2873.

11. Seo J-O, Han SI, and Lim S-C. Role of CDK8 and β-catenin in colorectal

adenocarcinoma. Oncology Reports (2010) 24, 285-291.

12. Adler AS, McCleland ML, Truong T et al. CDK8 maintains tumor de-differentiation

and embryonic stem cell pluripotency. Cancer Research (2012).

13. Kapoor A, Goldberg MS, Cumberland LK, Ratnakumar K, et al.

Table 1: Inhibitory Effect of Test Compounds on CDK8 activity

CDK8

Ex. Structure IUPAC name MW

ICso(nM)

1-[(3S)-1- ([l,2,4]triazolo[4,3-

V-NH b]pyridazin-6-

1 10.7 391 yl)pyrrolidin-3-yl]-3-[3- (trifluoromethyl)phenyl] ur

ea

CDK8

Ex. Structure IUPAC name MW

IC₅₀(nM)

(S)-l-(3-chloro-4-

HN— ((dimethylamino)methyl)p

16 henyl)-3-(l-(2- (methylamino)pyrimidin- 4-yl)pyrrolidin-3-yl)urea

1 -(4- Azetidin- 1 -ylmethyl-

— -NH 3-chloro-phenyl)-3-[(S)-l-

17 (2-methylamino- 2600 416 pyrimidin-4-yl)-pyrrolidin- 3-yl]-urea

l-[3-Chloro-4-(4-methyl-

— -NH piperazin- 1 -ylmethyl)-

18 phenyl]-3-[(S)-l-(2- 81 459 methylamino-pyrimidin-4- yl) -pyrrolidin-3 - yl] -urea

l-[3-Chloro-4-(4-ethyl-

— -NH

piperazin- 1 -ylmethyl)-

19 phenyl]-3-[(S)-l-(2- 53 473 methylamino-pyrimidin-4- yl) -pyrrolidin-3 - yl] -urea

l-(3-Chloro-4-morpholin-

— NH 4-ylmethyl-phenyl)-3-[(S)-

20 1 -(2-methylamino- 500 446 pyrimidin-4-yl)-pyrrolidin- 3-yl]-urea

l-(4-

Dimethylaminomethyl-3-

— -NH

trifluoromethyl-phenyl) - 3 -

21 35 437

[(S)- l-(2-methylamino- pyrimidin-4-yl)-pyrrolidin- 3-yl]-urea

While a number of embodiments have been described, these examples may be altered to provide other embodiments that utilize the compounds and methods described herein. Therefore, the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.

Claims

We claim:

A compound of formula

(I)

or a salt thereof, wherein:

1 is selected from:

and wherein R¹ is independently optionally substituted by 1, 2 or 3 R^x groups;

R 2" and R 3^J are independently H, Ci-^alkyl, C₂_₁₂alkenyl, C₂_₁₂alkynyl, carbocyclyl, aryl, heteroaryl, heterocyclyl, halo, -OR^a, -SR^a, -N(R^a)₂, -CN, -N0₂, -C(0)R^a, -C0₂R^a, -C(0)N(R^a)₂, -C(0)SR^a, -C(0)C(0)R^a, -C(0)CH₂C(0)R^a, -C(S)N(R^a)₂, -C(S)OR^a, -S(0)R^a, -S0₂R^a, -S0₂N(R^a)₂, -N(R^a)C(0)R^a, -N(R^a)C(0)N(R^a)₂, -N(R^a)S0₂ R^a,

-N(R^a)S0₂N(R^a)₂, -N(R^a)N(R^a)₂, -N(R^a)C(=N(R^a))N(R^a)₂, -C(=N)N(R^a)₂, -C=NOR^a, -C(=N(R^a))N(R^a)₂, -OC(0)R^a, or -OC(0)N(R^a)₂, wherein each Ci_i₂alk l, C₂_₁₂alkenyl,

C₂_₁₂alkynyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl of R 2 and R 3 are each independently optionally substituted with one or more groups R^x; or R¹ and R² taken together with the atoms to which they are attached form a 4, 5, 6, 7, or 8 membered carbocyclyl, heterocyclyl, heteroaryl or aryl, which carbocyclyl, heterocyclyl, heteroaryl and aryl is optionally substituted with one or more groups R^x;

each R^a and R^b are independently H, C^a cyl, C₂_₆alkenyl, C₂_₆alkynyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl, wherein each Ci^alkyl, C₃_₆alkenyl, C₃_₆alkynyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl is independently optionally substituted with one or more groups R^x; or R^a and R^b are taken together with the nitrogen to which they are attached to form a 3-6 membered heterocyle optionally substituted by oxo, halo, or C^alkyl optionally subsituted by oxo or halo; each R^v is independently hydrogen, C^alkyl, C₂_₆alkenyl, C₂_₆alkynyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl, wherein each Ci^alkyl, C₂_₆alkenyl, C₂_₆alkynyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl is optionally substituted with one or more groups independently selected from oxo, halo, amino, hydroxyl, and C^Ce alkyl that is optionally substituted with one or more groups independently selected from oxo and halo; or two R^v are taken together with the nitrogen to which they are attached to form a 3-6 membered heterocyclyl that is optionally substituted with one or more groups independently selected from oxo, halo and C^alkyl that is optionally substituted with one or more groups independently selected from oxo and halo; and

each R^x is independently oxo, Ci^alkyl, C₂_₆alkenyl, C₂_₆alkynyl, Ci^haloalkyl,

carbocyclyl, aryl, heteroaryl, heterocyclyl, -F, -CI, -Br, -I, -N0₂, -N(R^V)₂, -CN, -C(O)- N(R^V)₂, -S(0)-N(R^v)₂, -S(0)₂-N(R^v)₂, -0-R^v, -S-R^v, -0-C(0)-R^v, -0-C(0)-0-R^v, -C(O)- R^v, -C(0)-0-R^v, -S(0)-R^v, -S(0)₂-R^v, -0-C(0)-N(R^v)₂, -N(R^v)-C(0)-OR^v, -N(R^v)-C(0)- N(R^V)₂, -S(0)₂-N(R^v)₂, -N(R^v)-C(0)-R^v, -N(R^v)-S(0)-R^v, -N(R^v)-S(0)₂-R^v, -N(R^v)-S(0)- N(R^V)₂, or -N(R^v)-S(0)₂-N(R^v)₂, wherein any Ci^alkyl, C₂_₆alkenyl, C₂_₆alkynyl, Ci_ 6haloalkyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl is independently optionally substituted with one or more groups selected from oxo, halo, -N0₂, -N(R^V)₂, -CN,-C(0)- N(R^V)₂, -S(0)-N(R^v)₂, -S(0)₂-N(R^v)₂, -0-R^v, -S-R^v, -0-C(0)-R^v, -C(0)-R^v, -C(0)-0-R^v, - S(0)-R^v, -S(0)₂-R^v, -C(0)-N(R^v)₂, -S(0)₂-N(R^v)₂, -N(R^v)-C(0)-R^v, -N(R^v)-S(0)-R^v - N(R^v)-S(0)₂-R^v and C^alkyl that is optionally substituted with one or more groups independently selected from oxo and halo; and

wherein when R² is F and R³ is hydrogen, then R^x is other than methyl, ethyl, trifluoromethyl or 2-fluorophenyl.

2. The compound of claim 1 or a salt thereof, wherein

R¹ is selected from:

R² and R³ are independently H, Ci-^alkyl, halo, or -OR^a, wherein each Ci-^alkyl of R² and R³ are each independently optionally substituted with one or more groups R^x;

each R^a and R^b are independently H, C^a cyl, wherein each C^a cyl is independently

optionally substituted with one or more groups R^x; each R^v is independently C^alkyl; and

each R^x is independently heterocyclyl, -F, or -N(R^V)₂, wherein any heterocyclyl is

independently optionally substituted with one or more groups selected from C_halky!; and

wherein when R² is F and R³ is hydrogen, then R^x is other than methyl, ethyl, trifluoromethyl or 2-fluorophenyl.

3. The compound of claims 1-2 wherein R 2 and R 3 are independently halo,

trifluoromethyl, C^aUcyl or Ci^alkoxy, wherein said alkyl and alkoxy are independently optionally substituted by -N(R^V)₂ or heterocyclyl; wherein said heterocyclyl is optionally substituted by oxo, halo or C^alkyl optionally substituted by halo or oxo.

4. The compound of claims 1-3 wherein R is hydrogen, chloro, methyl, (4- methylpiperazin-l-yl)methyl, trifluoromethoxyl, difluoromethoxyl, 4-azetidin-l-ylmethyl, morpholin-4-ylmethyl, dimethylaminomethyl, pyrrolidin-l-ylmethyl or 4-ethylpiperizin-l- ylmethyl.

5. The compound of claims 1-4 wherein R is hydrogen, trifluoromethyl, fluoro or chloro.

6. The compound of claims 1-5 wherein R^a and R^b are independently hydrogen or Q_ ₆alkyl.

7. The compound of claims 1-6 wherein R^a is hydrogen.

8. The compound of claims 1-7 wherein R^b is methyl.

9. The compound of claims 1-8, selected from the group consisting of:

l-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl]-3-[3- (trifluoromethyl)phenyl]urea;

l-[4-chloro-3-(trifluoromethyl)phenyl]-3-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6- yl)pyrrolidin-3-yl]urea;

l-(3-chlorophenyl)-3-[(3S)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl]urea; l-[(3R)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl]-3-[3- (trifluoromethyl)phenyl]urea;

l-[4-chloro-3-(trifluoromethyl)phenyl]-3-[(3R)-l-([l,2,4]triazolo[4,3-b]pyridazin-6- yl)pyrrolidin-3-yl]urea;

l-(3-chlorophenyl)-3-[(3R)-l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl]urea; (S)-l-(l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl)-3-(4-((4-ethylpiperazin-l- yl)methyl)-3-(trifluoromethyl)phenyl)urea;

l-[(3S)-l-(2-aminopyrimidin-4-yl)pyrrolidin-3-yl]-3-(3-chloro-4-methyl-phenyl)urea; l-[(S)-l-(2-Amino-pyrimidin-4-yl)-pyrrolidin-3-yl]-3-(4-chloro-3-trifluoromethyl-phenyl)- urea;

l-[4-chloro-3-(trifluoromethyl)phenyl]-3-[(3S) ^

3- yl]urea;

l-(3-chloro-4-methyl-phenyl)-3-[(3S)-l-[2-(methylamino)pyrimidin-4-yl]pyrrolidin-3- yl]urea;

l-[(3S)-l-[2-(methylamino)pyrimidin-4-yl]pyrrolidin-3-yl]-3-[4- (trifluoromethoxy)phenyl]urea;

l-[4-(difluoromethoxy)phenyl]-3-[(3S)-l-[2-(methylamino)pyrimidin-4-yl]pyrrolidin-3- yl]urea;

(S)-l-(4-(azetidin-l-ylmethyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidm yl)urea;

(S)-l-(4-(azetidin-l-ylmethyl)-3-fluorophenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea;

(S)-l-(3-chloro-4-((dimethylamino)methyl)^^

yl)pyrrolidin-3-yl)urea;

(S)-l-(4-(azetidin-l-ylmethyl)-3-chlorophenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea;

(S)-l-(3-chloro-4-((4-methylpiperazin-l-yl)methyl)phenyl)-3-(l-(2-(methylamino)pyrim ^

4- yl)pyrrolidin-3-yl)urea;

(S)-l-(3-chloro-4-((4-ethylpiperazin-l-yl)methyl)phenyl)-3-(l-(2-(methylamino)pyrimidm yl)pyrrolidin-3-yl)urea;

(S)-l-(3-chloro-4-(morpholinomethyl)phenyl)-3-(l-(2-(methylamino)pyrimidin-4- yl)pyrrolidin-3-yl)urea; (S)-l-(4-((dimethylamino)methyl)-3-(trifluoromethyl)phenyl)-3-(l-(2- (methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea;

(S)-l-(4-(azetidin-l-ylmethyl)-3-(trifluoro

4-yl)pyrrolidin-3-yl)urea;

(S)-l-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)-3-(4-(pyrrolidin-l-ylmethyl)-3 (trifluoromethyl)phenyl)urea;

(S)-l-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)-3-(4-((4-methylpiperazin-l- yl)methyl)-3-(trifluoromethyl)phenyl)urea;

(S)-l-(4-((4-ethylpiperazin-l-yl)methyl)-3-(trifluoromethyl)phenyl)-3-(l-(2- (methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)urea;

(S)-l-(l-(2-(methylamino)pyrimidin-4-yl)pyrrolidin-3-yl)-3-(4-(morpholinomethyl)-3

(trifluoromethyl)phenyl)urea;

l-(4-Azetidin-l-ylmethyl-3-trifluoromethy

yl)-pyrrolidin-3-yl]-urea;

(S)-l-(l-([l,2,4]triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl)-3-(3-chloro-4- methylphenyl)urea; and

pharmaceutically acceptable salts thereof.

10. The compound of claims 1-9, selected from:

and salts thereof.

11. A pharmaceutical composition comprising a compound of claims 1-10 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant, carrier, or vehicle.

12. The use of a compound of claims 1-10 in therapy.

13. The use of a compound of claims 1-10 for the treatment of a hyperproliferative disorder.

14. The use of a compound of claims 1-10 in the manufacture of a medicament for the treatment of a disease associated with CDK8 activity.

15. The compound of claims 1 - 10 or a pharmaceutically acceptable salt thereof for use as therapeutic active substances.

16. The compound of claims 1 - 10 or a pharmaceutically acceptable salt thereof for use as therapeutic active substances for the treatment of a hyperproliferative disorder.

17. A method of treating a disease associated with CDK8 activity, comprising administering an therapeutically effective amount of a compound of claims 1-9, to a patient in need thereof.

18. The invention as hereinbefore described.