HK1116349B - Pyrrolopyrimidines useful as inhibitors of protein kinase - Google Patents

Description

Pyrrolopyrimidines useful as protein kinase inhibitors

Technical Field

The present invention relates to compounds useful as inhibitors of Janus kinases (JAKs). The invention also provides pharmaceutically acceptable compositions comprising the compounds of the invention and methods of using the compositions in the treatment of various disorders.

Background

Janus kinases (JAKs) are a family of tyrosine kinases consisting of JAK1, JAK2, JAK3 and TYK 2. JAKs play a crucial role in cytokine signaling. Downstream substrates of JAK family kinases include transcriptional signal sensing and activation (STAT) proteins. JAK/STAT signaling has been implicated in the mediation of many aberrant immune responses, such as allergy, asthma, autoimmune diseases, such as transplant rejection, rheumatoid arthritis, amyotrophic lateral sclerosis, and multiple sclerosis, and solid and hematologic malignancies, such as leukemias and lymphomas. JAK2 has also been implicated in myeloproliferative disorders, including polycythemia vera, essential thrombocythemia, chronic idiopathic myelofibrosis, myelogenous tissue deformation with myelofibrosis, chronic myelogenous leukemia, chronic myelomonocytic leukemia, chronic eosinophilic leukemia, hypereosinophilic syndrome, and systemic mast cell disease.

The serine/threonine kinase (ROCK) family of proteins that form Rho-associated coiled coils are the effectors of Ras-related small gtpase Rho. The ROCK family includes p160ROCK (ROCK-1), ROK alpha/Rho-kinase/ROCK-II, protein kinase PKN and citrate kinase. ROCK has been implicated in a variety of diseases and conditions, including hypertension, chronic obstructive pulmonary disease, cerebral vasospasm, coronary vasospasm, bronchial asthma, erectile dysfunction, glaucoma, vascular smooth muscle cell proliferation, myocardial hypertrophy, malignancy, ischemia/reperfusion-induced injury, endothelial dysfunction, crohn's disease and colitis, axonal outgrowth, raynaud's disease, angina, alzheimer's disease, atherosclerosis, cardiac hypertrophy, and peripheral vascular fibrosis.

Protein kinase a (PKA, also known as cAMP-dependent protein kinase) is a tetrameric holoenzyme that contains two catalytic subunits, combined with a homodimeric regulatory subunit that acts to inhibit the catalytic subunit. Upon binding to cAMP (enzyme activation), the catalytic subunit dissociates from the regulatory subunit to give an active serine/threonine kinase. Three isoforms of the catalytic subunit have been reported to date (C- α, C- β and C- γ), with C- α being the most widely studied, primarily due to its elevated expression in primary and metastatic melanomas. PKA has been shown to regulate many vital functions including energy metabolism, gene transcription, proliferation, differentiation, reproductive function, secretion, neuronal activity, memory, contractility and motility.

Therefore, there is an urgent need to develop compounds useful as inhibitors of protein kinases, including the JAK family, ROCK and PKA kinases. Specifically, there is a need to develop compounds useful as inhibitors of JAK2 and JAK 3.

Disclosure of Invention

It has now been found that the compounds of the present invention and pharmaceutically acceptable compositions thereof are effective as inhibitors of protein kinases, particularly of JAK family kinases. In certain embodiments, these compounds are effective as inhibitors of JAK3 protein kinases. These compounds have the general formula I:

or a pharmaceutically acceptable salt thereof, wherein R¹、R²、Z¹、Z²And Z³As defined below.

These compounds and pharmaceutically acceptable compositions thereof are useful in treating or lessening the severity of a variety of conditions, including allergic conditions such as asthma and atopic dermatitis, autoimmune diseases such as SLE lupus and psoriasis, conditions associated with organ transplantation, myeloproliferative disorders, hypertension, chronic obstructive pulmonary disease, and proliferative disorders such as melanoma.

Detailed description of the invention

Compounds and definitions

The compounds of the present invention include those generally described above, further illustrated as major classes, minor classes, and species disclosed herein. Unless otherwise indicated, the following definitions will apply. For the purposes of the present invention, the chemical elements will be according to the CAS version of the periodic Table of the elements, Handbook of chemistry and Physics, 75^thAnd Ed is identified. In addition, the general principles of Organic Chemistry are described in "Organic Chemistry", Thomas Sorrell, university science Books, Sausaltito: 1999, and "March's Advanced organic chemistry", 5^th Ed.，Ed.：Smith、M.B.and March、J.，John Wiley&Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

As described herein, the compounds of the invention may be optionally substituted with one or more substituents, such as those set forth in the summary above, or as exemplified by particular classes, subclasses, and species of the invention. It is to be understood that the phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted". In general, the term "substituted," whether preceded by the term "optionally," means that a hydrogen radical in a given structure is replaced with a radical that is designated as a substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and if more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at each position.

As used herein, when the term "optionally substituted" is followed by a list, the term denotes all groups in the list that may be subsequently substituted. For example, if X is halogen; optionally substituted C_1-3Alkyl or phenyl; x may be an optionally substituted alkyl group or an optionally substituted phenyl group. Likewise, if the term "optionally substituted" precedes a list, the term also refers to all substitutable groups in the previous list, unless otherwise indicated. For example, if X is halogen, C_1-3Alkyl or phenyl, wherein X is optionally J^XSubstituted, then C_1-3Both alkyl and phenyl groups may optionally be substituted by J^XAnd (4) substitution. As will be apparent to one of ordinary skill in the art, there will be NO such as H, halogen, NO₂、CN、NH₂OH or OCF₃Etc. since they are not substitutable groups.

Substituent combinations contemplated by the present invention are preferably those that form stable or chemically feasible compounds. The term "stable" as used herein means compounds that are substantially unchanged when subjected to the conditions used for their preparation, detection, preferably recovery, purification, and for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that remains substantially unchanged in the absence of moisture or other chemically reactive conditions at a temperature of 40 ℃ or less for at least one week.

As described herein, a bond drawn from a substituent to the center of one ring within a polycyclic ring system (as shown below) represents the substitution of the substituent at any substitutable position in any ring within the polycyclic ring system. For example, panel a represents a possible substitution in any of the positions shown in panel b.

Drawing a and b

This also applies to polycyclic systems fused with alternative ring systems (which will be represented by dashed lines). For example, in scheme c, X is an optional substituent for ring a and ring B.

FIG. c is a drawing

However, if two rings in a polycyclic ring system each have a different substituent drawn from the center of each ring, then each substituent represents only a substitution on the ring to which it is attached, unless otherwise specified. For example, in figure d, Y is only an optional substituent for ring a and X is only an optional substituent for ring B.

FIG. d

As used herein, the term "aliphatic" or "aliphatic radical" refers to a straight (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or that contains one or more units of unsaturation, or to a monocyclic or bicyclic hydrocarbon that is fully saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as a "carbocycle", "cycloaliphatic" or "cycloalkyl"), which has a single point of attachment to the remainder of the molecule. Unless otherwise specified, aliphatic groups contain 1-20 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms, and in other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched substituted or unsubstituted alkyl, alkenyl, or alkynyl groups. Further examples of aliphatic groups include methyl, ethyl, propyl, butyl, isopropyl, isobutyl, vinyl, and sec-butyl.

The term "cycloaliphatic" (or "carbocycle" or "cycloalkyl") denotes a monocyclic C₃-C₈Hydrocarbons or bicyclic radicals C₈-C₁₂A hydrocarbon, which is fully saturated or contains one or more units of unsaturation, but is not aromatic, which has a single point of attachment to the rest of the molecule, wherein any single ring in said bicyclic ring system is a 3-7 membered ring. Suitable cycloaliphatic groups include, but are not limited to, cycloalkyl, cycloalkenyl, and cycloalkynyl. Further examples of aliphatic groups include cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cycloheptenyl.

The term "heterocycle", "heterocyclyl", "heterocycloaliphatic", or "heterocyclic" as used herein, refers to a non-aromatic, monocyclic, bicyclic, or tricyclic ring system in which one or more ring members are independently selected heteroatoms. In some embodiments, a "heterocycle", "heterocyclyl", "heterocycloaliphatic", or "heterocyclic" group has three to fourteen ring members in which one or more ring members is a heteroatom independently selected from oxygen, sulfur, nitrogen, or phosphorus, and each ring in the system contains 3 to 7 ring members.

Further examples of heterocycles include, but are not limited to, the following monocyclic rings: 2-tetrahydrofuryl, 3-tetrahydrofuryl, 2-tetrahydrothienyl, 3-tetrahydrothienyl, 2-morpholino, 3-morpholino, 4-morpholino, 2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-tetrahydropyrpiperazinyl, 2-tetrahydropyrpiperazinyl, 3-tetrahydropyrpiperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolinyl, 3-morpholinyl, 2-piperidinyl, 3-morpholinyl, 4-piperidinyl, 2-pyrrolidinyl, 3-morpholino, 2-morpholinyl, 3-pyrrolidinyl, 4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, 5-imidazolidinyl, and the following bicyclic rings: 3-1H-benzimidazol-2-one, 3- (1-alkyl) -benzimidazol-2-one, indolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, benzothiophenyl, benzodithiane, and 1, 3-dihydro-imidazol-2-one.

The term "heteroatom" means one or more oxygen, sulfur, nitrogen, phosphorus or silicon (including any oxidized form of nitrogen, sulfur, phosphorus or silicon; quaternized forms of any basic nitrogen or heterocyclic ring substitutable nitrogen, e.g. N (as in 3, 4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺(as in N-substituted pyrrolidinyl)).

The term "unsaturated" as used herein means that the group has one or more units of unsaturation.

The term "alkoxy" or "thioalkyl" as used herein means an alkyl group, as defined above, attached to the bulk carbon chain through an oxygen ("alkoxy") or sulfur ("thioalkyl") atom.

The terms "haloalkyl", "haloalkenyl" and "haloalkoxy" denote alkyl, alkenyl or alkoxy groups, as the case may be, substituted with one or more halogen atoms. The term "halogen" denotes F, Cl, Br or I.

The term "aryl", used alone or as part of a larger group "aralkyl", "aralkoxy", or "aryloxyalkyl", denotes monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring". The term "aryl" also denotes a heteroaryl ring system as defined below. Examples of aryl rings would include phenyl, naphthyl, and heteroaryl as listed below.

The term "heteroaryl", used alone or as part of a larger group "heteroaralkyl" or "heteroarylalkoxy", denotes monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, at least one ring in the system contains one or more heteroatoms, and wherein each ring in the system contains 3 to 7 ring members. The term "heteroaryl" may be used interchangeably with the term "heteroaryl ring" or the term "heteroaromatic".

Further examples of heteroaryl rings include the following monocyclic rings: 2-furyl, 3-furyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoimidazolylAzolyl, 4-isoAzolyl, 5-isoAzolyl, 2-Azolyl, 4-Azolyl, 5-Oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g. 3-pyridazinyl), 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g. 5-tetrazolyl), triazolyl (e.g. 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl, pyrazolyl (e.g. 2-pyrazolyl), isothiazolyl, 1, 2, 3-Oxadiazolyl, 1, 2, 5-Oxadiazolyl, 1, 2, 4-Oxadiazolyl, 1, 2, 3-triazolyl, 1, 2, 3-thiadiazolyl, 1, 3, 4-thiadiazolyl, 1, 2,5-thiadiazolyl, pyrazinyl, 1, 3, 5-triazinyl, and the following bicyclic rings: benzimidazolyl, benzofuranyl, benzothienyl, indolyl (e.g., 2-indolyl), purinyl, quinolyl (e.g., 2-quinolyl, 3-quinolyl, 4-quinolyl), and isoquinolyl (e.g., 1-isoquinolyl, 3-isoquinolyl, or 4-isoquinolyl).

Aryl (including aralkyl, aralkoxy, aryloxyalkyl, and the like) or heteroaryl (including heteroaralkyl and heteroaralkoxy, and the like) may contain one or more substituents. Suitable substituents on the unsaturated carbon atom of an aryl or heteroaryl group are generally selected from the group consisting of^X、J^Q、J^RThose listed in the definitions of (a), halogen, -R °, -OR °, -SR °,1, 2-methylenedioxy, 1, 2-ethylenedioxy, phenyl (Ph) optionally substituted by R °, O (Ph) optionally substituted by R °, - (CH) optionally substituted by R °, -R₂)_1-2(Ph), -CH ═ CH (Ph), -NO, optionally substituted with R °₂、-CN、-N(R°)₂、-NR°C(O)R°、-NR°C(S)R°、-NR°C(O)N(R°)₂、-NR°C(S)N(R°)₂、-NR°CO₂R°、-NR°NR°C(O)R°、-NR°NR°C(O)N(R°)₂、-NR°NR°CO₂R°、-C(O)C(O)R°、-C(O)CH₂C(O)R°、-CO₂R°、-C(O)R°、-C(S)R°、-C(O)N(R°)₂、-C(S)N(R°)₂、-OC(O)N(R°)₂、-OC(O)R°、-C(O)N(OR°)R°、-C(NOR°)R°、-S(O)₂R°、-S(O)₃R°、-SO₂N(R°)₂、-S(O)R°、-NR°SO₂N(R°)₂、-NR°SO₂R°、-N(OR°)R°、-C(＝NH)-N(R°)₂Or- (CH)₂)_0-2NHC (O) R °, wherein each occurrence of R ° is independently selected from hydrogen, optionally substituted C_1-6Aliphatic radical, unsubstituted 5-6 membered heteroaryl or heterocycle, phenyl, -O (Ph) or-CH₂(Ph), or, although as defined above, two independent occurrences of R.cndot.on the same or different substituents, taken together with the atom to which each R.cndot.group is bonded, form a 5-8-membered heterocyclyl, aryl, or heteroaryl ring, or a 3-8-membered cycloalkyl ring, having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Aliphatic of R DEGOptional substituents on the radical being selected from NH₂、NH(C_1-4Aliphatic radical), N (C)_1-4Aliphatic radical)₂Halogen, C_1-4Aliphatic radical, OH, O (C)_1-4Aliphatic group), NO₂、CN、CO₂H、CO₂(C_1-4Aliphatic radical), O (halogeno C_1-4Aliphatic group) or halogeno C_1-4Aliphatic radical, in which R ° is each of the abovementioned C_1-4Aliphatic groups are unsubstituted.

The aliphatic or heteroaliphatic group or the non-aromatic heterocycle may contain one or more substituents. Suitable substituents on the saturated carbon atoms of the aliphatic or heteroaliphatic groups or of the non-aromatic heterocyclic rings are selected from those listed above for the aryl or heteroaryl unsaturated carbons, and additionally include the following groups: o, S, NNHR^*、＝NN(R^*)₂、＝NNHC(O)R^*、＝NNHCO₂(alkyl) ═ NNHSO₂(alkyl) or ═ NR^*Wherein each R is^*Independently selected from hydrogen or optionally substituted C_1-6An aliphatic group. R^*Is selected from NH₂、NH(C_1-4Aliphatic radical), N (C)_1-4Aliphatic radical)₂Halogen, C_1-4Aliphatic radical, OH, O (C)_1-4Aliphatic group), NO₂、CN、CO₂H、CO₂(C_1-4Aliphatic radical), O (halogeno C_1-4Aliphatic group) or halo (C)_1-4Aliphatic radical), wherein R^*Each of the above C_1-4Aliphatic groups are unsubstituted.

Optional substituents on non-aromatic heterocyclic nitrogen are included herein J^QAnd R⁷Those listed in the definitions of, -R⁺、-N(R⁺)₂、-C(O)R⁺、-CO₂R⁺、-C(O)C(O)R⁺、-C(O)CH₂C(O)R⁺、-SO₂R⁺、-SO₂N(R⁺)₂、-C(＝S)N(R⁺)₂、-C(＝NH)-N(R⁺)₂or-NR⁺SO₂R⁺(ii) a It is composed ofIn R⁺Is hydrogen, optionally substituted C_1-6An aliphatic group, an optionally substituted phenyl group, an optionally substituted-O (Ph), an optionally substituted-CH₂(Ph), optionally substituted- (CH)₂)_1-2(Ph), optionally substituted-CH ═ CH (Ph), or unsubstituted 5-6 membered heteroaryl or heterocyclic ring, having one to four heteroatoms independently selected from oxygen, nitrogen or sulfur, or R, although as defined above, independently occurs twice on the same substituent or on different substituents⁺And each R⁺The atoms to which the groups are bonded together form a 5-8-membered heterocyclyl, aryl or heteroaryl ring, or a 3-8-membered cycloalkyl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R⁺Is selected from NH or an optional substituent on the phenyl ring₂、NH(C_1-4Aliphatic radical), N (C)_1-4Aliphatic radical)₂Halogen, C_1-4Aliphatic radical, OH, O (C)_1-4Aliphatic group), NO₂、CN、CO₂H、CO₂(C_1-4Aliphatic radical), O (halogeno C_1-4Aliphatic group) or halo (C)_1-4Aliphatic radical), wherein R⁺Each of the above C_1-4Aliphatic groups are unsubstituted.

As noted above, in some embodiments, two independent occurrences of R ° (or R [ - ])⁺Or any other variable similarly defined herein) together with the atom to which each variable is bonded form a 5-8-membered heterocyclyl, aryl or heteroaryl ring or a 3-8-membered cycloalkyl ring, having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Two independent occurrences of R ° (or R [ - ])⁺Or any other variable similarly defined herein) along with the atom to which each variable is bonded include, but are not limited to, the following: a) two independent occurrences of R ° (or R [ - ])⁺Or any other variable similarly defined herein) to the same atom and together with that atom form a ring, e.g. N (R DEG)₂Wherein the two occurrences of R ° together with the nitrogen atom form piperidin-1-yl, piperazin-1-yl, or morpholin-4-yl; and b) two independent occurrences of R ° (or R °)⁺Or any other term defined similarly hereinThe variables) are bonded to different atoms and together with these atoms form a ring, e.g.Wherein the phenyl group is substituted with two occurrences of OR DEG, which together with the oxygen atom to which they are bonded form a fused 6-membered oxygen containing ring:it will be appreciated that two independent occurrences of R ° (or R [ - ])⁺Or any other variable similarly defined herein) may form a variety of other rings along with the atom to which each variable is bonded, and the above detailed examples are not intended to be limiting.

The alkyl or aliphatic chain may optionally be interrupted by another atom or group. This means that the methylene units of the alkyl or aliphatic chain are optionally replaced by said other atoms or groups. Examples of such atoms or groups would include, but are not limited to, -NR-, -O-, -S-, -CO₂-、-OC(O)-、-C(O)CO-、-C(O)-、-C(O)NR-、-C(＝N-CN)、-NRCO-、-NRC(O)O-、-SO₂NR-、-NRSO₂-、-NRC(O)NR-、-OC(O)NR-、-NRSO₂NR-, -SO-or-SO₂-, wherein R is as defined herein. Alternative substitutions, unless otherwise specified, produce chemically stable compounds. Optional interrupts may occur within the chain and at both ends of the chain; a connection point and an end. Two alternative substitutions may also be adjacent to each other within the chain. Unless otherwise specified, if a substitution or interruption occurs at a terminus, a substitute atom is bonded to the terminal H. For example, if-CH₂CH₂CH₃Optionally interrupted by-O-, the resulting compound may be-OCH₂CH₃、-CH₂OCH₃or-CH₂CH₂OH。

Unless otherwise specified, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure; for example, the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of these compounds are within the scope of the invention.

Unless otherwise specified, all tautomeric forms of the compounds of the invention are within the scope of the invention. In addition, unless otherwise specified, the structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, except that hydrogen is replaced by deuterium or tritium or carbon is replaced by¹³C-or¹⁴C-enriched carbon instead of compounds having the structure of the present invention are within the scope of the present invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

The present invention relates to compounds of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹is H, -NO₂、-CN、-OCF₃Halogen or amino; or C_1-6Aliphatic radical, C_3-7Cycloaliphatic radical, C_1-6Alkoxy or C_1-4Haloalkyl, optionally substituted with 0-10J^RSubstituted by groups;

R²is H, -NO₂、-CN、-OCF₃Halogen or amino; or C_1-6Aliphatic radical, C_3-7Cycloaliphatic radical, C_1-6Alkoxy or C_1-4Haloalkyl, optionally substituted with 0-10J^RSubstituted by groups;

Z¹is C_1-6Aliphatic radicals or C_3-10Cycloaliphatic radical, optionally substituted by 0 to 10J^ZSubstituted by groups; if Z is¹The bond between the two is a double bond, then Z¹Or may be ═ O, ═ NR or＝C(R)₂；

Z²Is H or halogen; or C_1-10Haloalkyl, C_1-4Haloalkoxy, Y, - (V)_n)-CN、-(V_n)-NO₂、-(V_n)-OH、-(V_n)-(C_1-6Aliphatic group), - (V)_n)-(C_3-12Heterocyclyl), - (V)_n)-(C_6-10Aryl), - (V)_n) - (5-to 10-membered heteroaryl) or- (V)_n)-(C_3-10Cycloaliphatic radical), optionally substituted by 0 to 10J^ZSubstituted by groups; or

Z¹And Z²Together with the carbon atom to which they are attached form ring Q;

Z³is H or C_1-6Alkyl, optionally substituted with 0-3J^ZSubstituted by groups; or

Z¹、Z²And Z³Together with the carbon atoms to which they are attached form a 6-14 membered saturated, partially saturated, or unsaturated bicyclic ring having 0-3 heteroatoms; wherein

If Z is¹The valence bond to C being a triple bond, then Z²Is absent; and is

If Z is¹The valence bond to C is a double or triple bond, then Z³Is absent;

q is a 3-8 membered saturated or partially saturated monocyclic ring having 0-3 heteroatoms selected from nitrogen, oxygen, or sulfur, wherein said Q is optionally and independently fused to Q¹Or Q²(ii) a Or condensed with Q¹And Q²(ii) a Wherein said Q is optionally substituted by 0-4J^QSubstituted by groups;

Q¹is a 3-8 membered saturated, partially saturated or unsaturated monocyclic ring having 0-3 heteroatoms selected from nitrogen, oxygen or sulfur, wherein said Q¹The radical being optionally substituted by 0 to 4J^QSubstituted by groups;

Q²is a 3-8 membered saturated, partially saturated or unsaturated monocyclic ring having0-3 heteroatoms selected from nitrogen, oxygen or sulfur, wherein said Q²The radical being optionally substituted by 0 to 4J^QSubstituted by groups;

r is H, optionally substituted C_1-6Aliphatic radical, C_3-10Cycloaliphatic radical, C_6-10Aryl, 5-14 membered heteroaryl or 5-14 membered heterocyclyl; or two R groups, taken together on the same or different substituents with the atom to which each R group is bonded, form an optionally substituted 3-14 membered saturated, partially unsaturated, or fully unsaturated monocyclic, bicyclic, or tricyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, wherein R is optionally substituted with 0-10J^RSubstituted by groups;

each J^QAnd J^ZThe substituents are independently selected from hydrogen, -OCF on unsaturated carbon atom₃、C_1-6Haloalkyl, N (R)₂OR, halogen, Y, - (V)_n)-CN、-(V_n)-NO₂、-(V_n)-OH、-(V_n)-(C_1-6Aliphatic group), - (C)_3-10Cycloaliphatic radical) -C (O) R, - (C)_3-10Cycloaliphatic radical) - (C)_3-12A heterocyclic group); - (V)_n)-(C_3-12Heterocyclyl), - (V)_n)-(C_6-10Aryl), - (V)_n) - (5-to 10-membered heteroaryl), - (V)_n)-(C_3-10A cycloaliphatic group); wherein each J^QAnd J^ZOptionally up to 10J^RSubstituted by groups;

each J^QAnd J^ZThe substituents are selected from those listed above for the unsaturated carbon on the saturated carbon atom and the following: o ═ NN (R)^a)₂、＝NNHC(O)R^a、＝NNHCO₂(C_1-4Alkyl) ═ NNHSO₂(C_1-4Alkyl) and ═ NR^aWherein each J^QAnd J^ZOptionally up to 10J^RSubstituted by groups;

each J^QAnd J^ZThe substituents being independently selected from hydrogen, Y, - (V) on the nitrogen atom_n)-CN、-(V_n)-NO₂、-(V_n)-OH、-(V_n)-(C_1-6Aliphatic group), - (C)_3-10Cycloaliphatic radical) -C (O) R, - (C)_3-10Cycloaliphatic radical) - (C)_3-12Heterocyclyl), - (V)_n)-(C_3-12Heterocyclyl), - (V)_n)-(C_6-10Aryl), - (V)_n) - (5-to 10-membered heteroaryl), - (V)_n)-(C_3-10A cycloaliphatic group); two of which are J^ZWith radicals having each J, on the same or different substituents^ZThe atoms to which the groups are bonded together may optionally form an optionally substituted 3-14 membered saturated, partially unsaturated or fully unsaturated monocyclic, bicyclic or tricyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur; wherein each J^QAnd J^ZOptionally up to 10J^RSubstituted by groups;

J^Rselected from halogen, -N (R)^b)₂、SR^b、OR^bOxo, C_1-4Haloalkoxy, C_1-4Haloalkyl, L, - (L)_n)-(C_1-6Alkyl), - (L)_n)-(C_3-12Heterocyclyl), - (L)_n)-(C_6-10Aryl), - (L)_n) - (5-to 10-membered heteroaryl), - (L)_n)-(C_3-10Cycloaliphatic group), - (L)_n)-NO₂、-(L_n)-CN、-(L_n)-OH、-CO₂R^b、-COR^b、-OC(O)R^b、-NC(O)R^b；

L is C_1-10Alkyl radicals in which up to three methylene units are replaced by-NR^b-、-O-、-S-、-CO₂-、-OC(O)-、-C(O)CO-、C(O)-、-C(O)NR^b-、-C(＝N-CN)、-NR^bCO-、-NR^bC(O)O-、-SO₂NR^b-、-NR^bSO₂-、-NR^bC(O)NR-、-OC(O)NR^b-、-NR^bSO₂NR^b-, -SO-or-SO₂-substituted;

v is C_1-10Aliphatic radicals in which up to three methylene units are replaced by G^VIn which G is substituted^VSelected from-NR-, -O-, -S-, -CO₂-、-OC(O)-、-C(O)CO-、-C(O)-、-C(O)NR-、-C(＝N-CN)、-NRCO-、-NRC(O)O-、-SO₂NR-、-NRSO₂-、-NRC(O)NR-、-OC(O)NR-、-NRSO₂NR-, -SO-or-SO₂-；

Y is C_1-10Aliphatic radicals in which up to three methylene units are replaced by G^YIn which G is substituted^YSelected from-NR-, -O-, -S-, -CO₂-、-OC(O)-、-C(O)CO-、-C(O)-、-C(O)NR-、-C(＝N-CN)、-NRCO-、-NRC(O)O-、-SO₂NR-、-NRSO₂-、-NRC(O)NR-、-OC(O)NR-、-NRSO₂NR-, -SO-or-SO₂-；

R^aIs hydrogen or C_1-6Aliphatic radical, optionally substituted by 0-3J^RSubstituted by groups;

R^bis hydrogen or unsubstituted C_1-6An aliphatic group;

n is 0 or 1;

the conditions are as follows:

if R is¹And R²Is H, Z²And Z³Is H, then Z¹Is not methyl;

if R is¹Is CH₃，R²Is H, then Z¹、Z²And Z³Are not all H;

if R is¹And R²Is H, Z²And Z³Is H, then Z¹Is not unsubstituted phenyl, 4-pyridyl or one of the structures shown below:

or

If R is¹And R²Is H, then Z¹And Z²Together not being-C ≡ C-CH₂CH₂COOH。

According to one embodiment of the invention, Z¹、Z²And Z³Together with the carbon atom to which they are attached form a bicyclic ring of formula I:

wherein

Q³Is a 3-8 membered saturated, unsaturated or partially saturated monocyclic ring;

q and Q³Each optionally and independently substituted by 0-4J^QAnd (4) substituting the group.

In one embodiment, Q³Is cyclopropyl, optionally substituted by 0-2J^QSubstituted by a group represented by formula II:

according to another embodiment of the invention, Z¹And Z²Together with the carbon atoms to which they are attached form a monocyclic, bicyclic or tricyclic ring of formula III:

wherein

Z¹¹Selected from C, N, O or S;

Z¹²selected from C, N, O or S;

q is a 3-8 membered saturated or partially saturated monocyclic ring, optionally fused to Q¹Or Q²；

Q¹And Q²Each independently is a 3-8 membered saturated, unsaturated or partially saturated monocyclic ring; q, Q¹And Q²Each independently containing up to three heteroatoms selected from O, N or S; m is 0 to 4 and independently according to Q, Q¹And Q²Selecting; and Z³Is H; or if C and Z are¹¹Is a double bond, then Z³Is absent.

In some embodiments, Z¹¹And Z¹²Each independently carbon.

In one embodiment, Q is C_3-7Monocyclic ring, Q¹And Q²Is absent.

In another embodiment, Q and Q¹Together form a fused 6-to 14-membered bicyclic ring, Q²Is absent.

In another embodiment, Q, Q¹And Q²Together form a fused 8-20 membered tricyclic ring.

In one embodiment of the invention, Z¹²Is carbon, Q, Q¹And optionally Q²The fused ring of formula IV:

q, Q therein¹And Q²Each independently and optionally containing

a)0-2 heteroatoms selected from O, N or S; and

b) 0-4J^QAnd (4) a substituent.

In one embodiment, ring Q and ring Q¹The hydrogen atom at the point of fusion between is in the cis configuration, as shown in formula V:

in another embodiment, ring Q and ring Q¹The hydrogen atom of the condensed point between is in trans configuration

In one embodiment, C-Z¹¹Is a single bond.

In another embodiment, C ═ Z¹¹Is a double bond.

In certain embodiments, ring Q contains up to two heteroatoms. In other embodiments, ring Q contains one heteroatom; in other embodiments, ring Q contains zero heteroatoms.

In one embodiment, Q contains two heteroatoms, each of which is independently selected from nitrogen, sulfur, or oxygen; nitrogen and sulfur are preferred; more preferably nitrogen. In some embodiments, both heteroatoms are nitrogen. In other embodiments, one is nitrogen and the other is sulfur. In some embodiments, one heteroatom is nitrogen and the other is oxygen. In other embodiments, one heteroatom is nitrogen and the other is sulfur.

In another embodiment, Q contains one heteroatom selected from O, N or S. In some embodiments, the heteroatom is oxygen; in other embodiments, the heteroatom is nitrogen; in other embodiments, the heteroatom is sulfur. In some embodiments, the sulfur is optionally substituted with 0, 1, or 2 oxo groups.

Examples of heterocyclic groups include piperidine, piperazine, morpholine, thiomorpholine and pyrrolidine.

In some embodiments, ring Q is a 5-7 membered cycloaliphatic. Examples of cycloaliphatic radicals include cyclohexane, cyclopentane, cyclohexene and cyclopentene.

In other embodiments, Q¹Is a 6-membered aryl or 5-6 membered heteroaryl ring. Examples of aryl or heteroaryl rings include phenyl, pyridine, pyrimidine, thiophene, thiazole, tetrazole, triazole, pyrrole, furan, anda pyrazole.

In some embodiments, Q¹Is a 3-7 membered cycloaliphatic ring. Examples of cycloaliphatic cyclic rings include cyclohexane, cyclopentane, cyclohexene, cyclopentene, cycloheptene, cycloheptane, cyclopropane, cyclobutane, cyclopropene, and cyclobutene.

In other embodiments, Q¹Is a 3-7 membered heterocyclic ring. Examples of heterocyclic groups include piperidine, piperazine, morpholine, thiomorpholine, pyrrolidine, homopiperidine, and homopiperazine.

In one embodiment, Q or Q-Q¹Is represented by the following structure:

and

wherein R is⁷And J^QAre each independently selected from hydrogen, Y, - (V)_n)-CN、-(V_n)-NO₂、-(V_n)-OH、-(V_n)-(C_1-6Aliphatic group), - (V)_n)-(C_3-12Heterocyclyl), - (V)_n)-(C_6-10Aryl), - (V)_n) - (5-to 10-membered heteroaryl), - (V)_n)-(C_3-10Cycloaliphatic radical) and- (C)_3-10Cycloaliphatic radical) - (C)_3-12A heterocyclic group);

wherein for each Q and Q¹For m is independently 0-3; and

each R⁷And J^QOptionally and independently by 0-10J^RAnd (4) substituting the group.

In one embodiment, m is 0, 1 or 2. In another embodiment, m is 1 or 2. In some embodiments, m is 0; in other embodiments, m is 1; in other embodiments, m is 2.

In some embodiments, J^QIs Y, - (V)¹ _n)-CN、-(V¹ _n)-NO₂、-(V¹ _n)-OH、-(V¹ _n)-(C_1-6Aliphatic group), - (V)¹ _n)-(C_3-12Heterocyclyl), - (V)¹ _n)-(C_6-10Aryl), - (V)¹ _n) - (5-to 10-membered heteroaryl), - (V)¹ _n)-(C_3-10Cycloaliphatic radical) or- (C)_3-10Cycloaliphatic radical) - (C)_3-12A heterocyclic group); wherein

V¹is-G^V-(X)_pWherein X is C_1-9Aliphatic radicals in which up to two methylene units are replaced by-NR-, -O-, -S-, -CO-₂-、-OC(O)-、-C(O)CO-、-C(O)-、-C(O)NR-、-C(＝N-CN)、-NRCO-、-NRC(O)O-、-SO₂NR-、-NRSO₂-、-NRC(O)NR-、-OC(O)NR-、-NRSO₂NR-, -SO-or-SO₂-substituted;

p is 0 or 1;

n is 0 or 1;

G^Vselected from C-O, C (═ O) NR, S (O)₂Or S (O); and

said J^QOptionally by 0-10J_RAnd (4) substituting the group.

In one embodiment, X is optionally substituted C_1-4An aliphatic group. In some embodiments, X is optionally substituted C_1-4An alkyl group. In some embodiments, X is optionally substituted C_1-2An alkyl group.

In some embodiments, n is 0. In other embodiments, n is 1. In certain embodiments, p is 0. In other embodiments, p is 1.

In some embodiments, J^QOptionally by 0-10J^RAnd (4) substituting the group. In some embodiments, 0-5J^RA group; in other embodiments, 0-3J^RA group; in other embodiments, 0-2J^RA group; in some embodiments, one J^RGroup, in certain embodiments, 0J^RA group.

In one embodiment, G^VIs C ═ O.

In another embodiment of the invention, R¹And R²Each independently of the others being H, halogen, C_1-4Alkyl or C_1-4An alkoxy group. In one embodiment, R¹And R²Each independently is H.

In some embodiments, R⁷Independently selected from Y, - (V)¹ _n)-CN、-(V¹ _n)-NO₂、-(V¹ _n)-OH、-(V¹ _n)-(C_1-6Aliphatic group), - (V)¹ _n)-(C_3-12Heterocyclyl), - (V)¹ _n)-(C_6-10Aryl), - (V)¹ _n) - (5-to 10-membered heteroaryl), - (V)¹ _n)-(C_3-10Cycloaliphatic radical) or- (C)_3-10Cycloaliphatic radical) - (C)_3-12A heterocyclic group); wherein

V¹is-G^V-(X)_pWherein X is C_1-9Aliphatic radicals in which up to two methylene units are replaced by-NR-, -O-, -S-, -CO-₂-、-OC(O)-、-C(O)CO-、-C(O)-、-C(O)NR-、-C(＝N-CN)、-NRCO-、-NRC(O)O-、-SO₂NR-、-NRSO₂-、-NRC(O)NR-、-OC(O)NR-、-NRSO₂NR-, -SO-or-SO₂-substituted;

p is 0 or 1;

n is 0 or 1; and

G^Vselected from C-O, C (═ O) NR, S (O)₂Or S (O).

Representative examples of compounds of formula I are described in table 1.

TABLE 1

Formula VI

Wherein A is selected from:

or

In another embodiment of the present invention, Z¹And Z²Not connected to form a ring, Z³Is H or absent.

In one embodiment, Z¹Is H or C_1-6Aliphatic radical, optionally substituted by 0 to 3J^ZAnd (4) substituting the group. In some embodiments, Z¹Is H.

In certain embodiments, C ≡ Z¹Is a triple bond, Z²And Z³Is absent.

In other embodiments, C ═ Z¹Is a double bond, Z³Is absent.

In certain embodiments, Z¹Is O; in other embodiments, Z¹Is CH₂。

In certain embodiments, Z²Is optionally substituted Y, - (V)_n)-(C_1-6Aliphatic group), - (V)_n)-(C_3-12Heterocyclyl), - (V)_n)-(C_6-10Aryl), - (V)_n) - (5-to 10-membered heteroaryl) or- (V)_n)-(C_3-10A cycloaliphatic group). In some embodiments, n is 0; in other embodiments, n is 1.

In other embodiments, Z²Is an optionally substituted 5-7 membered monocyclic ring selected from heterocyclyl, cycloaliphatic, aryl or heteroaryl; preferably a 5-7 membered fully or partially saturated monocyclic ring selected from heterocyclyl or cycloaliphatic; more preferably a 6 membered monocyclic ring, having 0-2 nitrogen atoms. In a preferred embodiment of the invention, Z²Is piperidine, optionally substituted by 0-3J^ZAnd (4) substituting the group.

In some embodiments, Z²Is optionally substituted- (V)_n)-(C_3-10Cycloaliphatic) where n is 0. In one embodiment, Z²Is a bicyclooctane ring. In another embodiment, Z²Is C_5-7A cycloaliphatic group. In other embodiments, Z²Is C_5-7A cycloalkyl group.

In one embodiment, J^ZIs halogen, CF₃Optionally substituted C_1-4Haloalkyl, - (V)¹ _n)-CN、-(V¹ _n)-NO₂、-(V¹ _n)-OH、Y、-(V¹ _n)-(C_3-12Heterocyclyl), - (V)¹ _n)-(C_6-10Aryl), - (V)¹ _n) - (5-to 10-membered heteroaryl), - (V)¹ _n)-(C_3-10Cycloaliphatic radical) or- (C)_3-10Cycloaliphatic radical) - (C)_3-12A heterocyclic group); wherein

V¹is-G^V-(X)_pWherein X is C_1-9Aliphatic radicals in which up to two methylene units are replaced by-NR-, -O-, -S-, -CO-₂-、-OC(O)-、-C(O)CO-、-C(O)-、-C(O)NR-、-C(＝N-CN)、-NRCO-、-NRC(O)O-、-SO₂NR-、-NRSO₂-、-NRC(O)NR-、-OC(O)NR-、-NRSO₂NR-, -SO-or-SO₂Replacing;

p is 0 or 1; and

G^Vselected from C-O, C (═ O) NR, S (O)₂Or S (O).

In some embodiments, X is C_1-5An aliphatic group. In certain preferred embodiments, X is C_1-5An alkyl group. In other preferred embodiments, X is C_1-2An alkyl group.

In some embodiments, G^VSelected from C-O, C (═ O) NR, S (O)₂Or S (O). In certain embodiments, G^VIs C ═ O. In other embodiments, G^VIs C (═ O) NR. In other embodiments, G^VIs S (O)₂Or S (O).

In some embodiments, J^ZIs halogen, CF₃CN, optionally substituted C_1-6Aliphatic radical, C_1-4Haloalkyl, - (C)_1-6Alkyl radical)_n-R^J、-(C_1-6Alkyl radical)_n-C(＝O)R^J、-(C_1-6Alkyl radical)_n-CON(R^b)R^J、-(C_1-6Alkyl radical)_n-N(R^b)R^J、-(C_1-6Alkyl radical)_n-OR^J、-(C_1-6Alkyl radical)_n-OCON(R^b)R^J、-(C_1-6Aliphatic radical)_n-S(O)N(R^b)R^J、-(C_1-6Aliphatic radical)_n-S(O)R^JOr- (C)_1-6Aliphatic radical)_n-NHC(O)R^J(ii) a Wherein

R^JIs C_1-6Aliphatic radical, C_3-12Heterocyclic group, C_6-10Aryl, 5-to 10-membered heteroaryl or C_3-10A cycloaliphatic group;

n is 0 or 1.

In other embodiments, J^ZIs halogen, OR^J、N(R^b)₂、CF₃CN, optionally substituted C_1-6Alkyl, - (C)_1-6Alkyl radical)_n-R^J、-C(＝O)(C_1-6Alkyl), -CON (R)^b)(C_1-6Alkyl), -OCON (R)^b)(C_1-6Alkyl), -S (O) N (R)^b)(C_1-6Alkyl), -S (O) (C)_1-6Alkyl), -NHC (O) C_1-6Alkyl, - (C)_1-6Alkyl) -CONH, - (C)_1-6Alkyl) -N (R)^b)₂、-(C_1-6Alkyl) -OCON (R)^b)R^J、-(C_1-6Aliphatic radical) -S (O) N (R)^b)(C_6-10Aryl), -N (R)^b)C(O)N(R^b)R^Jor-N (R)^b)C(O)R^b。

In other embodiments, J^ZIs halogen, OR, N (R)^b)₂、CF₃CN, optionally substituted C_1-6Alkyl, - (C)_1-6Alkyl radical)_n-R^J、C(＝O)(C_1-6Alkyl), CONH, - (C)_1-6Alkyl) -CONH, - (C)_1-6Alkyl) -N (R)^b)₂、-(C_1-6Alkyl) -OCON (R)^b)R^J、-(C_1-6Aliphatic radical) -S (O) N (R)^b)(C_6-10Aryl), -N (R)^b)C(O)N(R^b)₂or-N (R)^b)C(O)R^b。

In certain embodiments, R^JIs C_6-10Aryl or 5-10 membered heteroaryl. In other embodiments, R^JIs C_1-6Aliphatic radicals or C_3-10A cycloaliphatic group. In some embodiments, R^JIs C_1-6An aliphatic group. In other embodiments, R^JIs C_3-10A cycloaliphatic group.

In some embodiments, n is 1. In other embodiments, n is 0.

In certain embodiments, J^ZIs optionally substituted-C (═ O) (C)_1-6Alkyl), -C (═ O) CH₂CN or C_1-6An alkyl group.

Representative examples of compounds of formula I are set forth in table 2.

TABLE 2

Formula VII

Wherein A is selected from:

or

Representative examples of compounds of formula I are set forth in table 3:

TABLE 3

General synthetic methods:

the compounds of the invention can generally be prepared by methods known to those skilled in the art for analogous compounds or those depicted in the examples below. In general, example 1 depicts several methods for preparing functionalized quinoxalines.

While certain exemplary embodiments are depicted and described herein, it will be appreciated that the compounds of the present invention may be prepared according to the methods generally described above, using appropriate starting materials, by methods generally available to those of ordinary skill in the art.

All references provided in the synthetic schemes and examples are incorporated herein by reference. All abbreviations, symbols and conventions used herein are consistent with those used in the contemporary scientific literature. See, for example, Janet s.dodd, ed., The ACS Style Guide: amanal for Authors and Editors, 2nd ed., Washington, d.c.: american Chemical Society, 1997, the entire disclosure of which is incorporated herein by reference. In addition, the following definitions describe the terms and abbreviations used herein:

Ts-Cl-p-toluenesulfonyl chloride (toluenesulfonyl chloride)

DMF-dimethylformamide

Tf-triflate salt

LiHMDS-lithium hexamethyldisilazide

(Lithium hexamethyldisilazide)

dppf-1, 1' -bis (diphenylphosphino) -ferrocene

Ac-acetyl group

DME-1, 2-dimethoxyethane

atm-atmosphere

EDCI-1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride

DIEA-diisopropylethylamine

LiHMDS-lithium hexamethyldisilazane

(Lithium Hexamethyldisilazane)

THF-tetrahydrofuran

HEPES-4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid

Glu-glutamate salt

Tyr-tyrosine

ATP-adenosine triphosphate

Ph-phenyl

Me-methyl group

BSA-bovine serum Albumin

DTT-dithiothreitol

Scheme 1

Conditions are as follows: (a) Ts-Cl, K₂CO₃，DMF；(b)Tf₂O, lutidine or LiHMDS, PhNTf₂-78 ℃ to room temperature; (c) bis-pinacolatoborane (bispinacolatoborane), Pd (dppf)₂Cl₂KOAc then water; (d) pd (PPh)₃)₄KOAc, twoAlkane or DME, 100-; (f) LiOH or NaOMe; (g) Pd-C, H₂atm

Scheme 1 is a representative scheme for the preparation of compounds of the present invention. Compound 1 is commercially available and may be protected with a suitable protecting group (e.g. tosyl) such as t.w. greene& P.G.M Wutz，″Protective Groups in Organic Synthesis″，3^rd Edition，John Wiley &Sons, Inc (1999) produced compound 2. Boronic acids (5) may be prepared from the corresponding vinyl halides (4b) or vinyl triflates (4a), such as Comins, d.l.; dehghani, a. tetrahedron lett.1992, 33, 6299-; McMurry, j.e.; scott, w.j.tetrahedron lett, 1983, 24, 979; stang, p.j.; fisk, p.j.synthesis, 1980, 283; stang, p.j.; fisk, p.j.synthesis, 1979, 438; takagi, j.; takahashi, k.; ishiyama, t.; miyaura, n.j.am.chem.soc., 2002, 124, 8001 and references therein. In the presence of a suitable base, e.g. KOAc or Na₂CO₃Pd-mediated cross-coupling of N-protected halides (e.g. chlorides) (2) with boronic acids (3) gives compounds of type (6), such as a.suzuki, h.c. brown "Organic Synthesis Via Boranes; volume 3: suzuki Copling "Aldrich Chemical Company: milwaukee, WI, 2003 and references therein. Under basic conditions (e.g. LiOH)_(aq)Or NaOMe) to give (7). At H₂Hydrogenation with Pd-C under an atmosphere to give (8). (7) The Cyclopropanation of (9) is obtained as described by Reiser, Oliver "Cyclopropanation and other microorganisms of Palladium-Carbe (and Carbyne) complexes" Handbook of Organic Chemistry for Organic Synthesis (2002), 11561-1577.

Scheme 2

Conditions are as follows: (a) i: HCl-HNMeOMe, EDCI, DIEA; ii: MeMgBr; (b) LiHMDS, PhNTf2, THF, -78 deg.C; (c) bis-pinacolborane, Pd (dppf)₂Cl₂KOAc then water; (d) pd (PPh)₃)₄KOAc, twoAlkane or DME, 100-; (e) i: LiOH or NaOMe, ii: HCl,; (f) r ' COCl, R ' OCOCOCl or R ' NCO; DIEA or R' OOH; EDCI; and DIEA/wherein R ' COCl, R ' OCOCl and R ' NCO represent suitable acid chlorides, oxalyl chlorides and isocyanates, which are commercially available or can be prepared from commercially available starting materials.

Scheme 2 shows other methods for preparing compounds of the invention. In addition to acid chlorides, isocyanates and oxalyl chlorides, other compounds that react with amines can also be used to form P₁And (4) substitution. Examples include, but are not limited to, R '-halo, R' -tosyl, R '-mesyl, R' S (O)₂Cl、R’NS(O)₂Cl, R ' OH, R ' COOH and R ' CH₂-a halogen. -C ═ CH₂The compounds may optionally be converted to-C ═ O compounds using ozonolysis (g).

Scheme 3

Scheme 3 shows other methods for preparing compounds of the invention. Aryl bromides or iodides may be coupled to substituted terminal alkynes in the presence of Palladium, a base and CuI under Sonogashira coupling conditions (Sonogashira, Kenkichi. "Palladium-catalyzedalkynylation" instruments(s): Negishi, Ei-ichi. handbook of Organopaladium Chemistry for Organic Synthesis (2002), 1: 493-529. Publisher: John Wiley&Sons, inc., Hoboken, n.j.), yielding the product shown.Denotes suitable terminal alkynes, which are commercially available or can be prepared from commercially available starting materials.

While certain exemplary embodiments are depicted and described above and herein, it will be appreciated that the compounds of the invention can be prepared according to the methods generally described above, using appropriate starting materials, by methods generally available to those of ordinary skill in the art.

Use, formulation and administration

As discussed above, the present invention provides compounds that are inhibitors of protein kinases, including the JAK family, ROCK and PKA kinases, particularly JAK2 and JAK3 kinases, and thus are useful in the treatment of diseases, disorders and conditions including, but not limited to, immunodeficiency disorders, inflammatory diseases, allergic diseases, autoimmune diseases, proliferative disorders, immunologically-mediated diseases, respiratory disorders. Thus, in a further aspect of the invention, pharmaceutically acceptable compositions are provided, wherein these compositions comprise any of the compounds as described herein, and optionally a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents.

It will also be appreciated that certain compounds of the invention can be present in free form for use in therapy, or suitably as a pharmaceutically acceptable derivative thereof. According to the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or any other adduct or derivative capable of providing, directly or indirectly, a compound as described herein or a metabolite or residue thereof upon administration to a patient in need thereof.

The term "pharmaceutically acceptable salt" as used herein, means those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit/risk ratio. "pharmaceutically acceptable salt" means any non-toxic salt of a compound of the present invention.

Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts are described in detail, for example, in j.pharmaceutical Sciences, 1977, 66, 1-19, by s.m.berge et al, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptanoates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, embonate, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, Propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium and N⁺(C_1-4Alkyl radical)₄And (3) salt. The invention also encompasses quaternization of any basic nitrogen-containing group of the compounds as disclosed herein. By means of such quaternization, products which are soluble or dispersible in water or oil can be obtained. Representative alkali metals or basesThe earth metal salt includes sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include, when appropriate, non-toxic ammonium, quaternary ammonium and amine cations, generated using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates.

As noted above, the pharmaceutically acceptable compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, adjuvant, or vehicle, as used herein, including any and all solvents, diluents, or other liquid excipients, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, e.w. martin (Mack Publishing co., Easton, Pa., 1980) disclose various carriers for formulating pharmaceutically acceptable compositions and known techniques for their preparation. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, e.g., any other component that produces any undesirable biological effect or interacts in a deleterious manner with a pharmaceutically acceptable composition, its use is contemplated as falling within the scope of the present invention. Some examples of materials capable of serving as pharmaceutically acceptable carriers 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 or 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 silicon dioxide; magnesium trisilicate; polyvinylpyrrolidone; a polyacrylate; waxes; polyethylene-polypropylene oxide-block polymers; lanolin; sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; crushed tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; a phosphate buffer solution; and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preserving and anti-oxidizing agents may also be present in the composition, according to the judgment of the person skilled in the art.

In another aspect, there is provided a method of treating or lessening the severity of a proliferative disorder, a cardiac disorder, a neurodegenerative disorder, an autoimmune disorder, a condition associated with organ transplantation, an inflammatory disorder or an immunologically-mediated disorder, comprising administering to a subject in need thereof an effective amount of the compound or a pharmaceutically acceptable composition comprising the compound. In certain embodiments of the invention, an "effective amount" of a compound or pharmaceutically acceptable composition is an amount effective to treat or reduce the severity of a proliferative disorder, a cardiac disorder, a neurodegenerative disorder, an autoimmune disorder, a disorder associated with organ transplantation, an inflammatory disorder, or an immunologically-mediated disorder. The compounds and compositions according to the methods of the present invention can be administered in any amount and by any route of administration effective to treat or reduce the severity of a proliferative disorder, a cardiac disorder, a neurodegenerative disorder, an autoimmune disorder, a disorder associated with organ transplantation, an inflammatory disorder, or an immunologically-mediated disorder. The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the infection, the particular drug, the manner in which it is administered, and the like. The compounds of the present invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein denotes physically discrete pharmaceutical units, as appropriate for the patient to be treated. It will be understood, however, that the total daily amount of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dosage level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular compound employed; the specific composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration, the route of administration, and the rate of excretion of the particular compound employed; the duration of the treatment; drugs used in combination or concomitantly with the specific compound employed; and other factors well known in the medical arts. The term "patient" as used herein means an animal, preferably a mammal, most preferably a human.

The pharmaceutically acceptable compositions of the present invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments or drops), buccally, as an oral or nasal spray, etc., to humans and other animals, depending on the severity of the infection being treated. In certain embodiments, the compounds of the present invention may be administered orally or parenterally at a dosage level of from about 0.01mg/kg to about 50mg/kg, preferably from about 1mg/kg to about 25mg/kg, of the subject's body weight per day, one or more times a day, to achieve the desired therapeutic effect.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers 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 and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

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 non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable carriers 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 may be employed including synthetic mono-or di-glycerides. In addition, fatty acids, such as oleic acid, may be used in the preparation of injectables.

The 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 the compounds of the invention, it is often desirable to delay absorption of the compounds following subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material which is poorly water soluble. The rate of absorption of a compound depends on its rate of dissolution, which in turn may depend on crystal size and crystal form. Alternatively, delayed absorption of the parenterally administered compound form is achieved by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are prepared by forming a microencapsulated matrix of the compound in a biodegradable polymer, such as polylactide-polyglycolide. Depending on the ratio of compound to polymer and the nature of the particular polymer employed, the release rate of the compound can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations can also be prepared by entrapping the compound in liposomes or microemulsions which are compatible with body tissues.

Rectal or vaginal compositions are preferably suppositories which can be prepared by mixing the compounds of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity to release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier, for example sodium citrate or dicalcium phosphate, and/or a) fillers or extenders, for example starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, for example carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) wetting agents, for example glycerol, d) disintegrants, for example agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) dissolution retarders, for example paraffin, f) absorption accelerators, for example quaternary ammonium compounds, g) wetting agents, for example cetyl alcohol and glycerol monostearate, h) absorbents, for example kaolin and bentonite, and i) lubricants, for example talc, calcium stearate, sodium silicate, and the like, Magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft or hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as polymeric polyethylene glycols and the like. Solid dosage forms such as tablets, dragees, capsules, pills and granules can be provided with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as polymeric polyethylene glycols and the like.

The active compound may also be in microencapsulated form, containing one or more of the above-mentioned excipients. Solid dosage forms such as tablets, dragees, capsules, pills and granules can be provided with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, for example sucrose, lactose or starch. Such dosage forms may also contain, under normal circumstances, other substances in addition to inert diluents, such as tableting lubricants and other tableting aids, for example magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers, as appropriate. Ophthalmic formulations, ear drops and eye drops are also encompassed within the scope of the present invention. In addition, the present invention encompasses the use of transdermal patches, which have the added advantage of controlling the delivery of compounds to the body. Such dosage forms may be prepared by dissolving or dispersing the compound in the appropriate medium. Absorption enhancers may also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

As generally described above, the compounds of the invention are useful as inhibitors of protein kinases, particularly JAK family kinases, ROCK and PKA kinases. In a specific embodiment, the compounds and compositions of the invention are inhibitors of JAK2 and JAK 3. The compounds and compositions are useful for treating or lessening the severity of a disease, disorder or condition in which activation of a JAK family kinase, ROCK and/or PKA is implicated. In a particular embodiment, the compounds and compositions are useful for treating or lessening the severity of a disease, disorder or condition in which activation of JAK2 or JAK3 is implicated. When activation of JAK2, JAK3, ROCK or PKA is implicated in a particular disease, disorder or condition, the disease, disorder or condition may also be referred to as a "JAK 2-mediated disease", "JAK 3-mediated disease", "ROCK-mediated disease" or "PKA-mediated disease", respectively. Thus, in a further aspect, the invention provides a method of treating or lessening the severity of a disease, disorder or condition in which activation of a JAK family kinase, ROCK or PKA, particularly JAK2 or JAK3, is implicated in the disease state.

The activity of the compounds used in the present invention as inhibitors of JAK family kinases, ROCK or PKA, in particular JAK2 or JAK3, may be determined in vitro, in vivo or in cell lines. In vitro assays include determining the inhibition of phosphorylation activity or ATPase activity of activated JAK2, JAK3, ROCK or PKA. A selective in vitro assay may quantify the ability of an inhibitor to bind JAK2, JAK3, ROCK or PKA. Binding of the inhibitor can be measured by radiolabelling the inhibitor prior to binding, isolating the inhibitor/kinase complex and determining the amount of radiolabel bound. Alternatively, binding of the inhibitor may be determined by incubating the novel inhibitor with the relevant kinase bound to a known radioligand in a competitive assay.

The term "measurably inhibits" as used herein means that there is a measurable change in JAK2, JAK3, ROCK or PKA activity between a sample comprising the composition and JAK2, JAK3, ROCK or PKA and an equivalent sample comprising JAK2, JAK3, ROCK or PKA, respectively, without the presence of the composition.

The term "JAK 3-mediated disease" or "JAK 3-mediated disorder" as used herein means any disease or other deleterious disorder in which JAK3 is known to play a role. JAK 3-mediated conditions or diseases also represent those conditions or disorders that are alleviated by treatment with a JAK3 inhibitor. Such conditions include, without limitation, immune responses (e.g., allergic or type I hypersensitivity reactions), asthma, autoimmune diseases (e.g., transplant rejection, graft-host disease, rheumatoid arthritis, amyotrophic lateral sclerosis, and multiple sclerosis), neurodegenerative diseases (e.g., Familial Amyotrophic Lateral Sclerosis (FALS)), and solid and hematologic malignancies (e.g., leukemias and lymphomas).

The term "JAK 2-mediated disease" or "JAK 2-mediated disorder" as used herein means any disease or other deleterious disorder in which JAK2 is known to play a role. JAK 2-mediated conditions or diseases also represent those conditions or disorders that are alleviated by treatment with a JAK2 inhibitor. Such conditions include, without limitation, myeloproliferative disorders including polycythemia vera, essential thrombocythemia, chronic idiopathic myelofibrosis, myelogenous tissue deformation with myelofibrosis, chronic myelogenous leukemia, chronic myelomonocytic leukemia, chronic eosinophilic leukemia, hypereosinophilic syndrome, and systemic mastocytosis.

The term "ROCK-mediated disease" or "ROCK-mediated disorder" as used herein refers to any disease or other deleterious disorder in which ROCK is known to play a role. ROCK-mediated disorders or diseases also refer to those diseases or disorders 1 that are alleviated by treatment with a ROCK inhibitor. Such conditions include, without limitation, hypertension, angina pectoris, cerebrovascular contraction, asthma, peripheral circulation disorders, premature labor, cancer, erectile dysfunction, arteriosclerosis, spasms (cerebral vasospasm and coronary vasospasm), retinopathies (e.g., glaucoma), inflammatory disorders, autoimmune disorders, AIDS, osteoporosis, myocardial hypertrophy, ischemia/reperfusion-induced injury, endothelial dysfunction, alzheimer's disease or benign prostatic hyperplasia. In other embodiments, the conditions in which such known ROCKs play a role include, without limitation, hypertension, cerebral vasospasm, coronary vasospasm, bronchial asthma, preterm labor, erectile dysfunction, glaucoma, vascular smooth muscle cell proliferation, myocardial hypertrophy, malignancy, ischemia/reperfusion-induced injury, endothelial dysfunction, crohn's disease and colitis, axonal outgrowth, raynaud's disease, angina, alzheimer's disease, benign prostatic hyperplasia, or atherosclerosis.

The term "PKA-mediated disease" or "PKA-mediated disorder" as used herein means any disease or other deleterious disorder in which PKA is known to play a role. PKA-mediated conditions or diseases also mean those diseases or conditions which are alleviated by treatment with a PKA inhibitor. PKA-mediated diseases or disorders include, but are not limited to, proliferative disorders and cancer.

It will also be appreciated that the compounds and pharmaceutically acceptable compositions of the present invention may be used in combination therapy, that is, the compounds and pharmaceutically acceptable compositions may be administered simultaneously, prior to, or subsequent to one or more other desired therapeutic agents or pharmaceutical procedures. The particular combination of therapies (therapeutic agents or procedures) used in the combination regimen will take into account the compatibility of the desired therapeutic agent and/or procedure with the desired therapeutic effect to be achieved. It will also be appreciated that the therapies used may achieve the desired effect on the same condition (e.g., the compounds of the invention may be administered simultaneously with another drug used to treat the same condition), or they may achieve different effects (e.g., control of any side effects). As used herein, other therapeutic agents that are normally administered to treat or prevent a particular disease or condition are referred to as "appropriate for the disease or condition being treated.

The amount of the other therapeutic agent in the composition of the present invention will not exceed the amount normally administered in a composition containing the therapeutic agent as the only active ingredient. Preferably, the amount of the other therapeutic agent in the presently disclosed compositions will be about 50% to 100% of the content in typical compositions containing the drug as the sole therapeutically active ingredient.

The compounds of the present invention or pharmaceutically acceptable compositions thereof may also be incorporated into compositions for coating implantable medical devices, such as prostheses, prosthetic valves, vascular grafts, stents and catheters. Thus, the present invention, in another aspect, includes a composition for coating an implantable device comprising a compound of the present invention as generally described above and as described in classes and subclasses herein, and a carrier suitable for coating said implantable device. In another aspect, the present invention includes an implantable device coated with a composition comprising a compound of the present invention as generally described above and in classes and subclasses herein, and a carrier suitable for coating the implantable device.

Stent's impression of blood vessels (stents) has been used, for example, to overcome restenosis (restenosis of the vessel wall after injury). However, patients using stents or other implantable devices are at risk for clot formation or platelet activation. These undesirable effects can be prevented or mitigated by pre-coating the device with a pharmaceutically acceptable composition comprising a kinase inhibitor. Suitable coatings and general methods of making coated implantable devices are described in U.S. Pat. nos. 6,099,562, 5,886,026, and 5,304,121. The coating is typically a biocompatible polymeric material such as hydrogel polymers, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate copolymers and mixtures thereof. The coating may optionally be further covered with a surface layer of a suitable fluorosilicone, polysaccharide, polyethylene glycol, phospholipid, or combinations thereof to impart controlled release characteristics to the composition.

Another aspect of the invention relates to inhibiting JAK2, JAK3, ROCK or PKA activity in a biological sample or patient, the method comprising administering to or contacting the biological sample with a compound of formula I or a composition comprising the compound. The term "biological sample" as used herein is an ex vivo or in vitro sample, including without limitation cell cultures and extracts thereof; biopsy material obtained from a mammal or an extract thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

Inhibition of JAK2, JAK3, ROCK or PKA kinase activity in a biological sample can be used for a variety of purposes known to those skilled in the art. Examples of such purposes include, but are not limited to, blood transfusion, organ transplantation, biological sample storage, and biological assays.

Examples

For examples 1 to 7, 1H-NMR spectra were recorded at 500MHz using a Bruker AMX 500 instrument. Mass spectral samples were analyzed on a MicroMass ZQ or Quattro II mass spectrometer operating in single MS mode using electron jet ionization. The sample is introduced to the mass spectrometer using Flow Injection (FIA) or chromatographic techniques. All mobile phases of mass spectrometry consist of acetonitrile-water mixtures containing 0.2% formic acid as modifier. The term "R" as used herein_tt"indicates the HPLC retention time in minutes, which is related to the compound.

Example 1

4-chloro-7-tosyl-7H-pyrrolo [2, 3-d ] pyrimidine

Compound 1(307mg, 2.00mmol), tosyl chloride (418mg, 2.20mmol) and freshly ground K₂CO₃A slurry of (1.1g, 8.0mmol) DMF (5.0mL) was stirred at room temperature for 2 hr. The mixture was partitioned between water and EtOAc, the organic phase was washed with brine (2 ×), dried (Na)₂SO₄) Filtered and concentrated to give the title compound (583mg, 1.89mmol, 95% yield) as a white solid.

4-cyclopentenyl-7-tosyl-7H-pyrrolo [2, 3-d ] pyrimidine

4-chloro-7-tosyl-7H-pyrrolo [2, 3-pyrimidine (62mg, 0.20mmol), cyclopentenoboric acid (27mg, 0.24mmol), KOAc (78mg, 0.80mmol) and Pd (PPh)₃)₄(11mg, 0.010mmol) in bisThe mixture in alkane (0.6mL) was heated to 150 ℃ (MW, 600s) in a sealed tube. Subjecting the reaction mixture to flash chromatography (SiO)₂0-50% EtOAc-hexanes gradient elution) to give the title compound (55mg, 0.16mmol, 81% yield) as a white solid.

4-cyclopentenyl-7H-pyrrolo [2, 3-d ] pyrimidine (Compound 1)

A mixture of 4-cyclopentenyl-7-tosyl-7H-pyrrolo [2, 3-d ] pyrimidine (55mg, 0.16mmol) in methanol (0.5mL) was treated with NaOMe (0.5N, 0.25mL) and warmed to 60 ℃ for 25 min. The reaction was diluted, quenched with TFA, concentrated, and flash chromatographed to give the title compound (19mg) as a white solid.

LC-MS R_tt＝1.57min，(M+H⁺)186.00

¹H NMR(500MHz，CDCl3)9.30(brs，1H)，8.86(s，1H)，7.33(dd，1H)，6.98(dd，1H)，6.79(dd，1H)，3.06(m，2H)，2.70(m，2H)，2.13(q，2H)

Example 2

4-cyclopentyl-7H-pyrrolo [2, 3-d ] pyrimidine (Compound 3)

4-cyclopentenyl-7H-pyrrolo [2, 3-d ]]A mixture of pyrimidine (11mg, 0.060mmol) and Pd-C (10% palladium on carbon, 22mg) in EtOAc (1mL) in H₂Stirring under atmosphere (balloon) for 5 hr. The mixture was filtered and concentrated to give the title compound (10mg) as a white solid.

LC-MS R_tt＝1.37min，(M+H⁺)188.10

¹H NMR(500MHz，CDCl3)9.00(brs，1H)，8.80(s，1H)，7.24(buried dd，1H)，6.63(dd，1H)，3.56(q，2H)，2.13(m，2H)，2.05(m，2H)，1.92(m，2H)，1.75(m，2H)

The compounds of examples 3-7 were prepared according to scheme II.

Example 3

4- (1-Phenylvinyl) -7H-pyrrolo [2, 3-d ] pyrimidine (Compound 8)

LC-MS R_tt＝2min，(M+H⁺)221

¹H NMR 500MHz；DMSO-d6：12.6(brm，1H)，8.85(s，1)，7.53(m，1H)，7.4(m，5H)，6.03(m，3H)

Example 4

4- (4-fluorophenylethenyl) -7H-pyrrolo [2, 3-d ] pyrimidine (Compound 5)

LC-MS R_tt＝2min，(M+H⁺)239

¹H NMR：500MHz；DMSO-d6：13.8(brm，1H)，8.93(s，1)，8.14(d，1H)，7.92(dd，2H)，7.83(m，1H)，7.71(d，1H)，7，35(dd，2H)，7.27(m，1H)

Example 5

4- (4-Chlorophenyl) -7H-pyrrolo [2, 3-d ] pyrimidine (Compound 6)

LC-MS R_tt＝2.3min，(M+H⁺)255

¹H NMR：500MHz；DMSO-d6：12.7(brm，1H)，8.90(s，1)，8.10(d，1H)，7.90(d，2H)，7.79(m，2H)，7.54(d，2H)，7.22(m，1H)

Example 6

4- (4- (trifluoromethyl) styryl) -7H-pyrrolo [2, 3-d ] pyrimidine (Compound 7)

LC-MS R_tt＝2.7min，(M+H⁺)289

¹H NMR：500MHz；DMSO-d6：12.5(brm，1H)，8.88(s，1)，8.15(d，1H)，8.07(d，2H)，7.90(d，1H)，7.85(d，2H)，7.75(m，1H)，7.45(m，1H)，7.17(m，1H)

Example 7

4-styryl-7H-pyrrolo [2, 3-d ] pyrimidine (Compound 4)

LC-MS R_tt＝2min，(M+H⁺)221

¹H NMR：500MHz；DMSO-d6：12.8(brm，1H)，8.93(s，1)，8.14(d，2H)，7.85(d，2H)，7.83(m，1H)，7.75(d，1H)，7.51(d，2H)，7.47(m，1H)，7.28(m，1H)

Example 8

Scheme III

Step 1: compound A (1- (tert-butyloxycarbonyl) -3-piperidinecarboxylic acid) (4.60g, 20.0mmol) was suspended in 40mL CH₂Cl₂In (1). EDCI (4.60g, 24.0mmol) was added followed by N, O-dimethylamine (HCl) (2.34g, 24.0mmol) and catalytic DMAP. The resulting mixture was stirred at room temperature overnight. All volatiles were removed under reduced pressure. The residue was dissolved in saturated NaHCO₃Aqueous solution and EtOAc. The layers were separated and the organic layer was washed with brine and MgSO₄Dried, filtered and evaporated to dryness. The product was used as is without further purification. Yield 4.7g, about 86%. H NMR (500MHz, CDCl3)3.73(s, 3H), 3.18(s, 3H), 2.87-2.81(m, 4H), 1.94(s, H), 1.73-1.65(m, 4H), 1.49-1.46(m, 9H).

Step 2: at 0 ℃ N₂Next, to a solution of compound B (4.7g, 17.2mmol) in 70mL THF was added a solution of 3.0M (11.5mL, 34.5mmol) methylmagnesium bromide in THF. After the addition was complete, the cooling bath was removed and the resulting mixture was allowed to warm to room temperature and stirred overnight. Mixing the obtained mixture with saturated KHSO₄The aqueous solution was quenched and diluted with EtOAc. The organic phase was washed with brine, over MgSO₄Dried, filtered and evaporated to dryness. The crude product was passed through a plug of silica gel using 5-20% EtOAc in hexaneAnd (4) eluting with alkane. The yield was 2.37g, about 60%. H NMR (500MHz, CDCl3)4.10(d, J ═ 12.0Hz, H), 3.92(s, H), 2.94(dd, J ═ 10.3, 13.3Hz, H), 2.82-2.77(m, H), 2.52-2.48(m, H), 2.18(s, 3H), 1.98(dd, J ═ 3.6, 12.9Hz, H), 1.73-1.69(m, H), 1.56-1.44(m, 11H).

And step 3: compound C (2.37g, 10.4mmol) was dissolved in 5.0mL THF at-78 deg.C (IPA-dry ice bath) N₂Next, add to LiHMDS solution (13.0mL, 13.0 mmol). After 30min, 2- [ N, N-bis (trifluoromethanesulfonyl) amino group was added]Pyridine (4.11g, 11.5mmol), and after 10min the cooling bath was removed. The resulting mixture was gradually warmed to room temperature and stirred overnight. Mixing the obtained mixture with saturated KHSO₄The aqueous solution was quenched and diluted with EtOAc. The layers were separated and the organic layer was washed with brine and MgSO₄Dried, filtered and evaporated to dryness. The crude product was passed through a plug of silica gel eluting with 10% EtOAc in hexanes. The yield was 2.93g, about 79%. H NMR (500MHz, CDCl3)5.19(d, J ═ 4.1Hz, H), 5.01(dd, J ═ 1.0, 4.1Hz, H), 4.14-4.11(m, H), 2.84(dd, J ═ 9.7, 13.2Hz, 3H), 2.41(s, H), 2.04-2.00(m, H), 1.72(t, J ═ 3.4Hz, H), 1.52-1.46(m, 11H).

And 4: compound D (2.93g, 8.2mmol) was dissolved in 30mL of toluene. Bis (pinacolato) diboron (2.07g, 8.2mmol) was added followed by triphenylphosphine (117.3mg, 0.44mmol) and potassium phenoxide (1.48g, 11.2 mmol). The RM was degassed with Ar for 5 min. Trans-dichlorobis (triphenylphosphine) palladium (II) (157.0mg, 0.22mmol) was added and the resulting mixture was stirred at 55 ℃ for 3 h. The resulting mixture was allowed to cool to room temperature and stirred overnight. The resulting mixture was taken up in saturated NaHCO₃Aqueous and EtOAc dilution. The layers were separated and the organic layer was washed with brine and MgSO₄Dried, filtered and evaporated to dryness. The crude product was passed through a plug of silica gel eluting with 5-15% EtOAc in hexanes. Phenol was still present, so the product was dissolved in Et₂O, washing with 1N NaOH solution. The organic phase is passed over MgSO₄Dried, filtered and evaporated to dryness. The yield was 2.01g, about 73%. H NMR (500MHz, CDCl3)5.83(d, J ═ 2.6Hz, H), 5.65(s, H), 4.07(dd, J ═ 1.6, 12.8Hz, 2H), 2.64-2.58(m, 2H),2.26(t，J＝11.2Hz，H)，1.82-1.79(m，H)，1.67-1.64(m，H)，1.50(t，J＝3.7Hz，11H)，1.29-1.19(m，12H)。

and 5: compound E (45.7mg, 0.14mmol) was dissolved in 1.0mL DME. Adding 4-chloro-7H-pyrrolo [2, 3-d ] benzene]Pyrimidine (20.8mg, 0.14mmol), followed by 2.0MNa₂CO₃Solution (200. mu.L, 0.4 mmol). The resulting mixture was degassed with Ar for 5 minutes and catalytic tetrakis (triphenylphosphine) palladium (0) was added. The resulting mixture was warmed to 160 ℃ via microwave irradiation. After 10 minutes, the resulting mixture was cooled to room temperature. Subjecting the mixture to hydrogenation with hydrogen₂O and EtOAc dilution. The layers were separated and the organic layer was washed with brine and MgSO₄Dried, filtered and evaporated to dryness. The crude residue was chromatographed on silica gel, eluting with 10-30% EtOAc in hexanes. Yield 7.1mg of compound 22.

Example 9

Procedure IV

(a) Tri-n-butyl (1-ethoxyvinyl) tin, Pd (PPh)₃)₂Cl₂Toluene, 90 ℃ (b)6N HCl, MeOH, THF (c) HBr, HOAc (d) HNR₁R₂，Et₃N (e) NaOR or HOR, Et₃N (f) LiOH or NaOMe

4-iodo-7- (toluene-4-sulfonyl) -7H-pyrrolo [2, 3-d]Pyrimidine: adding [5g, 16.2mmol]4-chloro-7- (toluene-4-sulfonyl) -7H-pyrrolo [2, 3-d]Pyrimidine (as described in example 1) was added in small portions to 100mL of cold (0 ℃) stirred 47% stabilized hydroiodic acid and stirred for one hour with cooling; then the temperature was raised to ambient temperature and stirred furtherFor 5 hr. The reaction mixture was diluted with water and the solid was isolated via suction filtration and washed with additional water. The crude solid was dissolved in dichloromethane, washed twice with saturated sodium bicarbonate solution, washed with brine and dried (Na)₂SO₄) The solvent was removed under reduced pressure and triturated with a 2: 1 mixture of hexane/MTBE to give 5.7g of the white product (88%).¹H NMR: in CDCL3, 500Mhz is δ 8.61(s, 1H), 8.06(d, 2H J ═ 8.5Hz), 7.75(d, 1H J ═ 4.1Hz), 7.32(d, 2H J ═ 8.5Hz), 6.45(d, 1H J ═ 4.1Hz), 2.4(s, 3H).

Step a: 4- (1-ethoxy-vinyl) -7- (toluene-4-sulfonyl) -7H-pyrrolo [2, 3-d]Pyrimidine: mixing [10g, 25mmol]4-iodo-7- (toluene-4-sulfonyl) -7H-pyrrolo [2, 3-d]Pyrimidine (as described above) with bis-triphenylphosphine palladium (II) dichloride [2.0g, 2.85mmol]Dissolved/suspended together in 200mL of anhydrous toluene. The mixture was purged with nitrogen for-5 minutes, then heated to 90 ℃ in an oil bath under nitrogen atmosphere. Slowly dropwise over 2 hours [12.66mL, 13.54g, 37.5mmol]A solution of tri-n-butyl (1-ethoxyvinyl) tin in 100mL of anhydrous toluene. After the addition was complete, the mixture was heated under nitrogen for an additional 6 hours. The reaction was cooled to ambient temperature and the solvent was removed under reduced pressure until the remaining volume was 1/5. 160mL of petroleum ether was added to this slurry, the mixture was stirred for 1 hour, the solid was isolated via suction filtration and washed with petroleum ether. The moist solid was suspended in acetonitrile, stirred for one hour, the solid was re-isolated via suction filtration and air dried. The pale yellow solid obtained was 7.2g, representing an 82% yield and was used without further treatment.¹H NMR: in CDCL3, 500Mhz is δ 8.9(s, 1H), 8.07(d, 2H, J ═ 8.5Hz), 7.7(d, 1H, J ═ 4.1Hz), 7.28(d, 2H, J ═ 8.5Hz), 7.04(d, 1H, J ═ 4.1Hz), 5.7(d, 1H, J ═ 2Hz), 4.58(d, 1H, J ═ 2Hz), 4.0(quart, 2H), 2.4(s, 3H), 1.5(t, 3H).

Step b: 1- [7- (toluene-4-sulfonyl) -7H-pyrrolo [2, 3-d]Pyrimidin-4-yl]-an ethane ketone: the reaction solution is stirred until the solution is dissolved in water [7.25g, 21.12mmol]4- (1-ethoxy-vinyl) -7- (toluene-4-sulfonyl) -7H-pyrrolo [2, 3-d]The pyrimidine was dissolved in 50mL each of methanol and THF and stirred with 10mL of 6N HCl at ambient temperature for 4.0 hours. The solvent was removed under reduced pressure and the residue partitioned between dichloromethane and saturated sodium bicarbonate solution. The organic fraction was washed with brine, dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The crude product was triturated with a mixture of MTBE and petroleum ether (1: 4) for several hours, and finally the solid was isolated by suction filtration and air-dried. 5.95g of pale yellow product represents 89% yield and is used without further purification.¹H NMR：500Mhz CDCL3δ9.0(s，1H)，8.08(d，2H、J＝8.4Hz)，7.87(d，，1H、J＝4.1Hz)，7.3(m，3H)，2.8(s，3H)，2.4(s，3H)。

Step c: 2-bromo-1- [7- (toluene-4-sulfonyl) -7H-pyrrolo [2, 3-d]Pyrimidin-4-yl]-an ethane ketone: adding [5.95g, 18.88mmol]1- [7- (toluene-4-sulfonyl) -7H-pyrrolo [2, 3-d]Pyrimidin-4-yl]-Ethanone dissolved/suspended in 90mL of glacial acetic acid and [7.53mL, 10.197g, 37.76mmol]30% hydrogen bromide in acetic acid. To this stirred mixture was added dropwise [0.970mL, 3.02g, 18.88mmol ] at ambient temperature over 1.0 hour]Bromine in 10mL glacial acetic acid. The reaction mixture was stirred at ambient temperature for an additional 4.0 hours during which time a yellow precipitate formed. The solvent was removed under reduced pressure and the residue partitioned between dichloromethane and saturated sodium bicarbonate solution. The organic phase was washed with water, brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude solid was triturated/stirred with MTBE overnight via suctionThe solid was isolated by filtration and air dried to give 4.2g of a pale yellow solid in 56.6% yield.¹H NMR: in CDCL3, 500Mhz is δ 9.1(s, 1H), 8.09(d, 2H, J ═ 8.5Hz), 7.93(d, 1H, J ═ 4.0Hz), 7.33(d, 2H, J ═ 8.5Hz), 7.29(d, 1H, J ═ 4.0Hz), 4.83(s, 2H), 2.4(s, 3H).

Step d, e: the conversion of bromide can be accomplished by nucleophilic displacement with a primary or secondary amine under basic conditions (i.e., step d) or by treatment with an alcohol (i.e., step e).

Step f: the compound was deprotected as described for compound 1.

Example 10

Procedure V

(a) Alkylzinc halides, THF, RT-100C (b) LiOH/THF or NaOMe/MeOH

(a) Alkylzinc halides, THF, rt-80 c (b) NaOMe/MeOH steps a, b: 4-bicyclo [2.2.1]hept-2-yl-7H-pyrrolo [2, 3-d]Pyrimidine: to a solution of N-tosyl-4-chlorodiazepine (40mg, 0.13mmol) in anhydrous THF (0.5mL) was added the appropriate alkylzinc halide (e.g., exo-2-norbornyl zinc bromide [0.40mL, 0.5M)]) The mixture was stirred at room temperature overnight. The mixture was then heated to 80 ℃ for an additional 1.5hr and cooled to room temperature. The reaction mixture was treated with NaOMe (200uL, 0.5M) in MeOH at 50 deg.C for 1.5hr, then Rochelle's salt was added and the mixture was extracted repeatedly with EtOAc. The organic layer was filtered and concentrated. The crude residue was purified by HPLC to afford compound 26. LC-MS: RT ═ 1.86, (M + H) ═ 214.2;¹H NMR(DMSO)12.42(m，2H)，8.78(s，1H) 8.74(s, 1H), 7.62(brs, 2H), 6.82(s, 1H), 6.77(s, 1H), 3.76(m, 1H), 3.31(s, 1H), 2.75(m, 1H), 2.46(m, 1H), 2.37(m, 2H), 2.14(m, 2H), 1.90(m, 1H), 1.72(m, 1H), 1.67(d, 2H), 1.59(m, 2H), 1.55-1.42 (composite m, 3H), 1.33(m, 2H), 1.23(m, 1H), 1.17(d, 1H), 0.99(m, 1H).

Compounds 23, 24, 25, 27, 28, 29, 30 and 31 were prepared using the methods described above.

Table 4 below depicts exemplary of certain compounds of the present invention¹H-NMR data (NMR) and liquid chromatography data, reported as mass plus protons (M + H) and Retention Time (RT) determined from electron injection, wherein the compound numbers in table 4 correspond to the compounds depicted in table 3 (blank boxes mean no test performed):

TABLE 4

Compound #	M+H+	LC-MS Rt	1H NMR
				2	Lot 1：200.00	Lot 1：1.93	Lot 1: (500MHz, CDCl3)8.92(brs, 1H), 8.81(s, 1H), 7.26 (buried dd, 1H), 6.86(m, 1H), 6.72(dd, 1H), 2.70(m, 2H), 2.34(m, 2H), 1.84(m, 2H), 1.76(m, 2H)
9	301.00	2.05	(CDCl3)10.07(brs，1H)，8.86(s，1H)，7.35(d，1H)，6.83(d，1H)，6.73(brs，1H)，4.24(brs，2H)，3.71(brs，2H)，2.86(brs，2H)，1.52(s，9H)

Compound #	M+H+	LC-MS Rt	1H NMR
				10			(d 4-methanol) 8.71(s, 1H), 7.52(d, 1H), 6.86(m, 1H), 6.84(d, 1H), 4.01(brs, 2H), 3.54(dd, 2H), 3.08(brs, 2H)
11	213.80	2.40	(500MHz，CDCl3)9.24(s，1H)，7.7(brs，1H)，7.56(s，1H)，7.02(d，1H)，6.93(brs，1H)，3.68(m，2H)，2.62(m，2H)，2.28(m，1H)，2.16(m，1H)，2.03(m，1H)，1.94(m，1H)，1.29(d，3H)
				12	228.19	2.17	(500MHz, CD3OD)8.90(s, 1H), 7.81(d, 1H), 6.95(d, 1H), 5.83(brs, 2H), 2.79(m, 1H), 1.85 (composite m, 4H), 1.75(brd, 1H), 1.47-1.24 (composite m, 5H)
13	218.00	1.50	500Mhz；DMSO-d6：12.1(brs，1H)，8.7(s，1H)，7.5(s，1H)，7.0(s，1H)，6.73(s，1H)，3.45(s，2H)，2.9(s，4H)
				14	146.00	0.60	(CDCl3)10.70(brs，1H)，8.89(s，1H)，7.39(d，1H)，7.15(dd，1H)，6.73(d，1H)，6.66(dd，1H)，5.82(dd，1H)
15	242.90	0.52
				16	310.90	1.61
17	268.20	0.60	(d 4-methanol) 8.76(s, 1H), 7.61(d, 1), 6.93(d, 1H), 6.90 and 6.86(2m, 1H), 4.40 and 4.34(2m, 2H), 4.01 and 3.97(2s, 2H), 3.90 and 3.78(2t, 2H), 2.94(m, 2H)
				18	214.23	1.97	(500MHz, CD3OD)8.81(s, 1H), 7.66(d, 1H), 6.85(d, 1H), 5.78 and 5.77(twos, 2H), 1.94(m, 2H), 1.78(m, 2H), 1.70(m, 2H), 1.50(m, 2H) ppm.
19	214.21	1.75	(500MHz，CD3OD)8.86(s，1H)，7.83(d，1H)，7.08(d，1H)，7.05(m，1H)，3.16(m，1H)，2.99(m，1H)，2.33(m，1H)，2.20(m，1H)，1.23(d，3H)，1.18(d，3H)ppm.
				20	214.20	2.60	1H NMR(500MHz，CD3OD)8.86(s，1H)，7.84(m，1H)，

Compound #	M+H+	LC-MS Rt	1H NMR
							7.00(m，2H)，2.88(m，2H)，2.58(m，2H)，1.97(m，2H)，1.81(m，2H)，1.72(m，2H).
21	211.90	1.60	500MHz，CDCl3：12.3.0(brm，1H)，9.0(s，1H)，7.63(s，1H)，7.58(s，1H)，6.93(s，1H)，3.75(s，1H)，3.30(s，1H)，1.97(m，2H)，1.74(d，1H)，1.45(d，1H)，1.33(m，1H)，1.22(m，1H)
				22	328.90	2.46	500Mhz，CDCl3 10.16(s，br，1H)，8.87(s，1H)，7.34(s，1H)，6.67(s，1H)，5.59(s，1H)，4.11(d，br，2H)，3.18(m，1H)，2.79(t，2H)，2.00(m，1H)，1.71(m，2H)，1.64-1.48(m，2H)，1.43(s，9H).
23	224.20	1.91
				24	220.20	1.52	12.47(brs，1H)，8.79(s，1H)，7.65(s，1H)，6.81(s，1H)，3.55(s，3H)，3.39(q，1H)，3.16(m，2H)，1.15(d，3H)
25	176.20	1.65
				27	216.20	2.04
28	201.20	1.48
				29	234.20	1.52
30	243.20	1.91
				31	190.20	1.91

Example 11: assay for inhibition of JAK3

Screening for their ability to inhibit JAK using the following assaysA compound (I) is provided. The reaction was carried out in a kinase buffer containing 100mM HEPES (pH7.4), 1mM DTT, 10mM MgCl₂25mM NaCl and 0.01% BSA.

The substrate concentrations in the assay were 5. mu.M ATP (200. mu. Ci/. mu.mol ATP) and 1. mu.M poly (Glu)₄Tyr. The reaction was carried out at 25 ℃ and 1nM JAK 3.

To each well of a 96-well polycarbonate plate, 1.5. mu.l of candidate JAK3 inhibitor and 50. mu.l of kinase buffer containing 2. mu.M poly (Glu)₄Tyr and 10. mu. MATP. Then mixed, and the reaction was started by adding 50. mu.l of kinase buffer containing 2nM JAK3 enzyme. After 20 minutes at room temperature (25 ℃), the reaction was stopped with 50. mu.l of 20% trichloroacetic acid (TCA) also containing 0.4mM ATP. The entire contents of each well were then transferred to a 96-well glass fiber filter plate using a TomTek cell harvester. After washing, 60. mu.l scintillation fluid was added and the assay was performed on a Perkin Elmer TopCount³³And (3) binding of P.

Example 12: assay for inhibition of JAK2

As described above in example 11, but using the JAK-2 enzyme, final poly (Glu)₄Tyr concentration was 15. mu.M, and final ATP concentration was 12. mu.M.

Example 13: ROCK inhibition assay

Compounds were screened for their ability to inhibit ROCK I (AA 6-553) using a standard ligase assay (Fox et al, Protein Sci, 7, 2249, 1998). The reaction was carried out in a solution containing 100mM HEPES (pH7.5), 10mM MgCl₂25mM NaCl, 2mM DTT and 1.5% DMSO. The final substrate concentration in the assay was 45. mu.M ATP (Sigmachemical, St Louis, Mo.) and 200. mu.M Peptide (American Peptide, Sunnyvale, Calif.). The reaction was carried out at 30 ℃ and 45nM ROCK I. The final concentrations of the components of the conjugated enzyme system were 2.5mM phosphoenolpyruvate, 350. mu.M ADH, 30. mu.M/ml pyruvate kinase and 10. mu.g/ml lactate dehydrogenase.

Assay needle using standard coupling enzymeScreening the compound for its ability to inhibit ROCK. The assay was carried out in a solution containing 100mM HEPES (pH7.5), 10mM MgCl₂25mM NaCl, 2mM DTT and 1.5% DMSO. The final substrate concentration in the assay was 13. mu.M [ gamma-³³P]ATP(25mCi ³³P ATP/mmol ATP, Perkin Elmer, Cambridge, MA/SigmaChemicals, StLouis, MO) and 27. mu.M Myelin Basic Protein (MBP). The final enzyme concentration in the assay was 5nM ROCK. The measurement was performed at room temperature. 1.5. mu.l DMSO stock containing serial dilutions of a compound of the invention (concentrations ranging from 10. mu.M to 2.6nM) were placed in 96-well plates. To the plate, 50. mu.l of solution 1(100mM HEPES (pH7.5), 10mM MgCl₂，26mM[γ³³P]ATP). 50 μ l of solution 2(100mM HEPES (pH7.5), 10mM MgCl was added₂4mM DTT, 54mM MBP and 10nM ROCK). After 2 hours, the reaction was quenched with 50 μ l 30% trichloroacetic acid (TCA, Fisher) containing 9mM ATP. Mu.l of the quenched reaction was transferred to a glass fiber filter plate (Corning, Cat. No.3511) followed by 3 washes with 5% TCA. Add 50. mu.l Optima Gold scintillation fluid (Perkin Elmer) and Count plates on Top Count (Perkin Elmer). All data points were averaged to remove background and K was calculated using Prism software_i(app)。

Example 14: assay for inhibition of PKA

Compounds were screened for their ability to inhibit PKA using a standard coupled enzyme assay (Fox et al, Protein Sci, 1998, 7, 2249). In 100mM HEPES (pH7.5), 10mM MgCl₂25mM NaCl, 1mM DTT in a mixture with 3% DMSO. The final substrate concentrations in the assay were 50 μ M ATP (Sigma Chemical) and 80 μ M Peptide (Kemptide, American Peptide, Sunnyvale, Calif.). The reaction was carried out at 30 ℃ and 18nM PKA. The final concentrations of the components of the coupled enzyme system were 2.5mM phosphoenolpyruvate, 300. mu.M NADH, 30. mu.g/ml pyruvate kinase and 10. mu.g/ml lactate dehydrogenase.

An assay stock buffer solution was prepared containing all of the above reagents, with the exception of ATP and the test compound of the invention. Store 55. mu.lThe solution was placed in a 96-well plate followed by the addition of 2 μ l dmso stock containing serial dilutions of the test compounds of the invention (usually starting at a final concentration of 5 μ M). The plates were preincubated at 30 ℃ for 10 minutes and 5. mu.l ATP was added to prime the reaction (final concentration 50. mu.M). Initial reaction rates were determined over 15 minutes using a Molecular Devices SpectraMax Plus plate reader. IC was calculated from nonlinear regression analysis using the Prism Software package (GraphPad Prism version 3.0a for Macintosh, GraphPad Software, San Diego California, USA)₅₀And K_iAnd (4) data.

Table 5 depicts enzyme inhibition data (K) for certain exemplary compounds_i). The compound numbers in table 5 correspond to those depicted in table 3. In Table 5, "A" represents K of less than 0.5. mu.M_iAnd "B" represents a K between 0.5 and 5.0. mu.M_iAnd "C" represents a K of more than 5.0. mu.M_i。

TABLE 5

Compound #	JAK2	JAK3	PKA	ROCK
					1	A	A	B	B
2	A	A	B	B
					3	B	B	B	B
4	A	A	B	B
					5	A	A	B	B
6	A	A	B	B
					7	A	A	B	B
8	C	B	B	B
					9	A	A	B	B
10	C	C	B	B
					11	B	A	B	B
12	B	B	B	B
					13	A	A	B	B
14	B	B	B	B
					15	C	C	B	B
16	B	C	B	B
					17	A	B	B	B
18	A	A	B	B
					19	A	A	B	B
20	A	A	B	B
					21	A	A	B	B
22	B	B	B	B
					23	B	B	B	B
24	C	C	B	B
					25	B	B	B	B
26	A	A	B	B
					27	A	A	B	B
28	B	B	B	B
					29	B	B	B	B
30	B	B	B	B
					31	A	A	B	B

Claims (25)

1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

R¹is H;

R²is H;

Z¹is C_1-6Aliphatic radicals or C_3-10Cycloaliphatic radicals, optionally substituted by 0-10 number J^ZSubstituted by groups; if Z is¹The bond between the two is a double bond, then Z¹Or may be ═ C (R)₂；

Z²Is- (C)_1-6Aliphatic group), - (C)_3-12Heterocyclyl), - (C)_6-10Aryl) or- (C)_3-10Cycloaliphatic radical), optionally substituted by 0 to 10J^Z(iii) substituted with a group wherein the heterocyclyl is piperidine, piperazine, morpholine, thiomorpholine or pyrrolidine; or

Z¹And Z²Together with the carbon atom to which they are attached form ring Q;

Z³is H or C_1-6Alkyl, optionally substituted with 0-3J^ZSubstituted by groups; or

Z¹、Z²And Z³Together with the carbon atoms to which they are attached form a 6-14 membered saturated or partially saturated bicyclic ring;

wherein Z¹The valence bond to C is a single or double bond;

if Z is¹The bond between the two is a double bond, then Z³Is absent;

q is a 3-8 membered saturated or partially saturated monocyclic ring having 0-3 heteroatoms selected from nitrogen, oxygen, or sulfur; wherein said Q is optionally substituted by 0-4J^QSubstituted by groups;

r is H, C_1-6Aliphatic radical, C_3-10Cycloaliphatic radicals or C_6-10An aryl group; wherein each R is optionally substituted with 0-10J^RSubstituted by groups;

each J^QAnd J^ZThe substituents are independently selected from hydrogen, -OCF on unsaturated carbon atom₃、C_1-6Haloalkyl, N (R)₂OR, halogen, -CN, -NO₂、-OH、-(C_1-6Aliphatic group), - (C)_3-10Cycloaliphatic radical) -C (O) R, - (C)_3-10Cycloaliphatic radical) - (C)_3-12Heterocyclyl), - (C)_3-12Heterocyclyl), - (C)_6-10Aryl), - (C)_3-10A cycloaliphatic group), wherein the heterocyclic group is piperidine, piperazine, morpholine, thiomorpholine, or pyrrolidine; wherein each J^QOptionally up to 10J^RSubstituted by groups;

each J^QAnd J^ZThe substituents are selected from those listed above for the unsaturated carbon on the saturated carbon atom and the following: o ═ NN (R)^a)₂、＝NNHC(O)R^a、＝NNHCO₂(C_1-4Alkyl) ═ NNHSO₂(C_1-4Alkyl) and ═ NR^aWherein each J^QOptionally up to 10J^RSubstituted by groups;

each J^QAnd J^ZThe substituents being independently selected from hydrogen, -CN, -NO on the nitrogen atom₂、-OH、-(C_1-6Aliphatic group), - (C)_3-10Cycloaliphatic radical) -C (O) R, - (C)_3-10Cycloaliphatic radical) - (C)_3-12Heterocyclyl), - (C)_3-12Heterocyclyl), - (C)_6-10Aryl), - (C)_3-10A cycloaliphatic group), wherein the heterocyclic group is piperidine, piperazine, morpholine, thiomorpholine, or pyrrolidine; wherein each J^QOptionally up to 10J^RSubstituted by groups;

J^Rselected from halogen, -N (R)^b)₂、SR^b、OR^bOxo, C_1-4Haloalkoxy, C_1-4Haloalkyl, - (C)_1-6Alkyl), - (C)_3-12Heterocyclyl), - (C)_6-10Aryl), - (C)_3-10Cycloaliphatic group), -NO₂、-CN、-OH、-CO₂R^b、-COR^b、-OC(O)R^b、-NHC(O)R^bWherein the heterocyclic group is piperidine, piperazine, morpholine, thiomorpholine or pyrrolidine;

R^ais hydrogen or C_1-6An aliphatic group;

R^bis hydrogen or unsubstituted C_1-6An aliphatic group.

2. A compound according to claim 1, wherein Z¹And Z²Together with the carbon atom to which they are attached, form a compound of formula IV:

wherein

Z¹¹Is selected from C;

Z¹²selected from C, N, O or S;

q is a 3-8 membered saturated or partially saturated monocyclic ring, Q¹And Q²Is absent;

each Q independently contains up to three heteroatoms selected from O, N or S;

m is 0-4 and is independently selected based on Q; and

Z³is H; or if C and Z are¹¹Is a double bond, then Z³Is absent.

3. The compound according to claim 1, wherein Q is selected from

Wherein

R⁷And J^QEach independently selected from hydrogen, -CN, -NO₂、-OH、-(C_1-6Aliphatic group), - (C)_3-12Heterocyclyl), -C)_6-10Aryl), - (C)_3-10Cycloaliphatic radical) and- (C)_3-10Cycloaliphatic radical) - (C)_3-12Heterocyclyl), wherein the heterocyclyl is piperidine, piperazine, morpholine, thiomorpholine, or pyrrolidine;

m is independently 0-3 for each Q; and

each R⁷And J^QOptionally and independently by 0-10J^RAnd (4) substituting the group.

4. A compound of formula VII:

formula VII

Wherein A is selected from:

5. a compound according to claim 1, wherein Z¹And Z²Not connected to form a ring, Z³Is H or absent.

6. A compound according to claim 5, wherein Z¹Is H or C_1-6Aliphatic radical, optionally substituted by 0 to 3J^ZAnd (4) substituting the group.

7. A compound of formula VIII:

of the formula VIII

Wherein A is selected from:

8. a compound selected from the following table:

9. a composition comprising an effective amount of a compound of any one of claims 1-8 and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

10. Use of a compound of claim 1 for the preparation of a medicament for inhibiting JAK3 kinase activity in a patient.

11. Use of a compound according to claim 1 for the preparation of an agent that inhibits JAK3 kinase activity in a biological sample.

12. Use of a compound according to claim 1 for the manufacture of a medicament for the treatment or lessening the severity of a disease or condition selected from the group consisting of: a proliferative disorder, a cardiac disorder, a neurodegenerative disorder, an autoimmune disorder, a disorder associated with organ transplantation, an inflammatory disorder, or an immunologically-mediated disorder.

13. The use of claim 12, wherein the disease or disorder is asthma.

14. The use of claim 12, wherein the disease or disorder is transplant rejection.

15. Use of a compound according to claim 1 for the preparation of a medicament for inhibiting JAK2 kinase activity in a patient.

16. Use of a compound according to claim 1 for the preparation of an agent that inhibits JAK2 kinase activity in a biological sample.

17. Use of a compound according to claim 1 for the manufacture of a medicament for treating or lessening the severity of a myeloproliferative disorder in a patient in need thereof.

18. The use according to claim 17, wherein the myeloproliferative disorder is polycythemia vera, essential thrombocythemia, chronic spontaneous myelofibrosis, myelogenous tissue deformation with myelofibrosis, chronic myelogenous leukemia, chronic myelomonocytic leukocytes, chronic eosinophilic leukemia, hypereosinophilic syndrome, or systemic mastocytosis.

19. Use of a compound according to claim 1 for the preparation of a medicament for inhibiting ROCK kinase activity in a patient.

20. Use of a compound according to claim 1 for the preparation of an agent that inhibits ROCK kinase activity in a biological sample.

21. Use of a compound according to claim 1 for the manufacture of a medicament for the treatment or lessening the severity of a disease, disorder or condition selected from the group consisting of: a proliferative disorder, a cardiac disorder, a neurodegenerative disorder, a psychiatric disorder, an autoimmune disorder, a disorder associated with organ transplantation, an inflammatory disorder, an immunologically-mediated disorder, a viral disease, or a bone disorder.

22. Use of a compound according to claim 1 for the manufacture of a medicament for inhibiting PKA kinase activity in a patient.

23. Use of a compound according to claim 1 for the preparation of a formulation for inhibiting PKA kinase activity in a biological sample.

24. Use of a compound according to claim 1 for the manufacture of a medicament for treating or lessening the severity of a proliferative disorder in a patient in need thereof.

25. The use according to claim 24, wherein the proliferative disorder is cancer.