WO2017063103A1

WO2017063103A1 - Novel inhibitors and probes for kinases and uses thereof

Info

Publication number: WO2017063103A1
Application number: PCT/CN2015/000691
Authority: WO
Inventors: Zhengying Pan; Yingying ZUO; Xitao LI
Original assignee: Peking University Shenzhen Graduate School; Beijing Reciproca Pharmaceuticals Co. Ltd.
Priority date: 2015-10-12
Filing date: 2015-10-12
Publication date: 2017-04-20
Also published as: CN108779078B; CN108779078A

Abstract

The present invention relates to 2,5-diaminopyrimidine-based Bruton's tyrosine kinase inhibitors. The present invention also relates to 2,5-diaminopyrimidine-based affinity probes for Bruton's tyrosine kinase and uses of such probes for measuring the activity of Btk, for assessing the activity of modulators of Btk, and for assessing the pharmacokinetic and pharmacodynamic properties of such modulators.

Description

Novel Inhibitors and Probes for Kinases and Uses Thereof

FIELD OF INVENTION

The present invention relates to 2， 5-diaminopyrimidine-based Bruton’s tyrosine kinase inhibitors. The present invention also relates to 2， 5-diaminopyrimidine-based affinity probes for Bruton’s tyrosine kinase and uses of such probes for measuring the activity of Btk， for assessing the activity of modulators of Btk， and for assessing the pharmacokinetic and pharmacodynamic properties of such modulators.

BACKGROUND

Bruton’s tyrosine kinase (Btk) is a cytosolic non-tyrosine kinase that is expressed only in hematopoietic cells， except in natural killer and T cells. Btk participates in several signaling pathways， particularly in the B cell receptor (BCR) pathway， which is crucial in B-cell development and differentiation [Mohamed， A. J. et al. Bruton′s tyrosine kinase (Btk) ： function， regulation， and transformation with special emphasis on the PH domain. Immunol. Rev. 228， 58-73 (2009) ] .

In cells， Btk is activated by its upstream kinases through the phosphorylation of a tyrosine residue (Tyr551) ， followed by the autophosphorylation of another tyrosine residue (Tyr223) . The fully activated Btk then phosphorylates its substrates， including PLC-γ2 in the BCR pathway.

Extensive in vivo and clinical studies strongly suggest that Btk is involved in the development of multiple B-cell malignancies and autoimmune diseases such as rheumatoid arthritis and lupus [Rickert， R. C. New insights into pre-BCR and BCR signalling with relevance to B cell malignancies. Nat. Rev. Immunol. 13， 578-591 (2013) ] .

Multiple Btk inhibitors have been developed (Fig. 1) .

Ibrntinib (CRA-032765， PCI-32765，

) ， a covalent irreversible inhibitor from Celera/Pharmacyclics/Janssen， became the first clinically approved Btk-targeting drug in November 2013 [Pan， Z. et al. Discovery of selective irreversible inhibitors for Bruton′s tyrosine kinase. ChemMedChem. 2， 58-61 (2007) ] . CC-292 (AVL-292) from Celgene is the second covalent irreversible inhibitor that is currently undergoing clinical trials [Singh， J. et al. inventors； Celgene Avilomics Research， Inc.， assignee. 2， 4-Diaminopyrimidines useful as kinase inhibitors. United States patent US 8,609,679. 2013 Dec 17] .

Both ibrutinib and CC-292 form a covalent bond with a cysteine residue (Cys481) located at the rim of the ATP-binding pocket in Btk.

Other clinical-stage Btk inhibitors include a compound from ONO Pharmaceutical and PRN1008/HM71224 from Hanmi Pharmaceutical [Yamamoto， S. &Yoshizawa T. inventors； Ono Pharmaceutical Co.， Ltd.， assignee. Purinone derivative. United States patent US 8,940,725. 2015 Jan 27； Hanmi Pharmaceutical Company Limited， Safety， PK/PD， Food Effect Study of Orally Administered HM71224 in Healthy Adult Male Volunteers. Available at： https： //clinicaltrials. gov/ct2/show/NCT01765478 (Accessed： 4th July 2015) ] .

GDC-0834， a non-covalent reversible Btk inhibitor from Gilead/Roche， was evaluated in a Phase I clinical trial， but no recent developments have been reported [Liu， L. et al. Significant species difference in amide hydrolysis of GDC-0834， a novel potent and selective Bruton′s tyrosine kinase inhibitor. Drug Metab. Dispos. 39， 1840-1849 (2011) ] .

Target engagement refers to the occupancy of intended biological targets by drug molecules [Copeland， R. A.， Pompliano， D. L. &Meek， T. D. Drug-target residence time and its implications for lead optimization. Nat. Rev. Drug Discov. 5， 730-739 (2006) ] . This information is crucial for building a correlation between phenotypic observations and inhibitor-biomolecule interactions at the molecular level. Targeted covalent drugs， due to their inherent reactive groups， are particularly suitable for developing small molecule affinity probes that may be used to measure the extent of target occupancy [Potashman， M. H. &Duggan， M. E. Covalent modifiers： an orthogonal approach to drug design. J. Med. Chem. 52， 1231-1246 (2009) ； Singh， J.， Petter， R. C.， Baillie， T. A. &Whitty， A. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 10， 307-317 (2011) ] .

PCI-33380 was designed based on the ibrutinib scaffold and has been used in both cellular and in vivo studies that demonstrated the connection between the inhibitor binding event and phenotypic readouts of cellular responses due to the inhibition of Btk functions [Honigberg， L. A. et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc. Natl. Acad. Sci. U. S. A. 107， 13075-13080 (2010) ] .

Furthermore， the use of fluorescent probes in clinical trials has played an important role in determining the appropriate dosage of drugs for patients [O′Brien， S. et al. Ibrutinib as initial therapy for elderly patients with chronic lymphocytic leukaemia or small lymphocytic lymphoma： an open-label， multicentre， phase 1b/2 trial. Lancet Oncol. 15， 48-58 (2014) ] .

In addition to PCI-33380， other fluorescent probes for Btk that also utilize the ibrutinib scaffold have been recently reported for the imaging of Btk in live cells (Fig. 2) [Turetsky， A.， Kim， E.， Kohler， R. H.， Miller， M. A. &Weissleder， R. Single cell imaging of Bruton′s tyrosine kinase using an irreversible inhibitor. Sci. Rep. 4， 4782 (2014) ； Zhang， Q.， Liu， H. &Pan， Z. A general approach for the development of fluorogenic probes suitable for no-wash imaging of kinases in live cells. Chem. Comm. 50， 15319-15322 (2014) ] .

As depicted in Fig. 3， affinity probes normally include three components： a recognition group， a reactive group and a reporting group. The recognition group directs the probe into the binding pocket of the targeted protein and facilitates the formation of a covalent bond between the reactive group and the biomolecule. The reporting group provides a convenient means of identifying probe-bound proteins within complex proteomes.

Fig. 4 shows a general scheme of assays to examine the target engagement of drug molecules. By sequentially adding inhibitors and probes into biological samples (cells， tissues， etc. ) ， the intensities of probe-labeled bands will give a direct readout of those biological targets are not occupied by inhibitors. As the concentration of inhibitors increases， a decrease of band intensity indicates a portion of biological targets are engaged by inhibitors.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a novel series of Btk covalent inhibitors based on the 2， 5-diaminopyrimidine scaffold. Another object of the present invention is to develop that series of inhibitors into a novel affinity Btk probe. The resulting probe can selectively label Btk and provide an efficient method of directly measuring the target engagement of Btk inhibitors in live cells.

In one aspect is a compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) ， or a pharmaceutically acceptable salt thereof：

wherein R is selected from the group consisting of a bond， carbonylalkyleneamino (e.g.， carbonylC_1-6alkyleneamino) ， ( ( (azacycloalk-2-yl) -alkyl) oxomethane) -l， N-diyl (e.g. ( ( (C_5-7azacycloalk-2-yl) -C_0-2alkyl) oxomethane) -1， N-diyl) ； preferably， R is selected from the group consisting of

and

wherein R₁ is selected from the group consisting of alkyl， arylalkyl， hydroxyalkyl， aminocarbonylalkyl， carboxylalkyl， aminoalkyl and heteroarylalkyl； preferably R₁ is selected from the group consisting of

and

more preferably， R₁ is selected from the group consisting of

and

wherein

p is 0， 1， 2 or 3， preferably 0；

q is 0， 1， 2 or 3， preferably 0；

R₁₁ is a substituent containing an end group selected from OH， COOH， CONH₂， NH₂， and a nitrogen containing heterocycle； preferably R₁₁ is an alkyl group substituted by a substituent selected from the group consisting of OH， COOH， CONH₂， NH₂， and a nitrogen containing heterocycle as end group， wherein one or more CH₂ moieties in said alkyl group are optionally replaced with a divalent group selected from the group consisting of-NH-， -CO-， -SO₂-and -SO-； more preferably， R₁₁ is selected from the group consisting of

and

In one aspect is the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) ， or a pharmaceutically acceptable salt thereof as the Btk inhibitor.

In one aspect is the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) ， or a pharmaceutically acceptable salt thereof as the Btk inhibitor for treating B-cell malignancies and autoimmune diseases such as rheumatoid arthritis and lupus.

Another aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) ， or a pharmaceutically acceptable salt thereof as the Btk inhibitor， in combination with one or more pharmaceutically acceptable carriers.

In one aspect is a Btk affinity probe， which comprises a Btk inhibitor moiety， a reporter moiety， and a linker moiety that links the Btk inhibitor moiety to the reporter moiety； wherein the Btk inhibitor moiety is derivable from the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) ， or a pharmaceutically acceptable salt thereof.

In one aspect is the use of the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) ， or a pharmaceutically acceptable salt thereof as a part of a Btk affinity probe， which comprises a Btk inhibitor moiety， a reporter moiety， and a linker moiety that links the Btk inhibitor moiety to the reporter moiety； wherein the Btk inhibitor moiety is derivable from the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) ， or a pharmaceutically acceptable salt thereof.

In one aspect is a Btk affinity probe as represented by the following formula (Ic) ：

wherein the Btk inhibitor moiety is derivable from the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) ， or a pharmaceutically acceptable salt thereof；

wherein X and Y are independently selected from the group consisting of a bond， -O (CO) -， -NR^a (CO) -， -NR^a-，

-O-， -S-， -S-S-， -O-NR^a-， -O (CO) O-， -O (CO) NR^a-， -NR^a (CO) NR^a-， N＝CR^a-， -S (CO) -， -S (O) -， and -S (O) ₂-；

wherein

forms a N-containing heterocycle；

R^a is hydrogen or alkyl.

In one embodiment is a Btk affinity probe wherein the linker moiety covalently links the Btk inhibitor moiety to the reporter moiety. In another embodiment is a Btk affinity probe wherein the Btk inhibitor moiety modifies a cysteine residue of a Btk enzyme. In a further embodiment is a Btk affinity probe wherein the Btk inhibitor moiety covalently modifies the cysteine residue of the Btk enzyme. In yet a further embodiment is a Btk affinity probe wherein the cysteine residue is in the ATP binding pocket of the Btk enzyme. In yet another embodiment is a Btk affinity probe wherein the cysteine residue is Cys 481 of the Btk enzyme. In one embodiment is a Btk affinity probe wherein the linker moiety is selected from a bond， an optionally substituted alkyl moiety， an optionally substituted heterocycle moiety， an optionally substituted amide moiety， a ketone moiety， an optionally substituted carbamate moiety， an ester moiety， or a combination thereof. In another embodiment is a Btk affinity probe wherein the linker moiety is a bond.

Also described herein is a Btk affinity probe wherein the reporter moiety is selected from the group consisting of a label， a dye， a photocrosslinker， a cytotoxic compound， a drug， an affinity label， a photoaffinity label， a reactive compound， an antibody or antibody fragment， a biomaterial， a nanoparticle， a spin label， a fluorophore， a metal-containing moiety， a radioactive moiety， a novel functional group， a group that covalently or noncovalently interacts with other molecules， a photocaged moiety， an actinic radiation excitable moiety， a ligand， a photoisomerizable moiety， biotin， a biotin analogue， a moiety incorporating a heavy atom， a chemically cleavable group， a photocleavable group， a redox-active agent， an isotopically labeled moiety， a biophysical probe， a phosphorescent group， a chemiluminescent group， an electron dense group， a magnetic group， an intercalating group， a chromophore， an energy transfer agent， a biologically active agent， a detectable label， or a combination thereof.

In another embodiment is a Btk affinity probe wherein the reporter moiety is a fluorophore. In yet another embodiment is a Btk affinity probe wherein the fluorophore is a Bodipy fluorophore. In yet a further embodiment is a Btk affinity probe wherein the Bodipy fluorophore is a Bodipy FL fluorophore.

Presented herein is a Btk affinity probe wherein the Btk inhibitor moiety is derived from an irreversible inhibitor of Btk. In one embodiment is a Btk affinity probe wherein the irreversible inhibitor of Btk is：

In another embodiment is a Btk affinity probe having the structure：

In a further embodiment is a Btk affinity probe wherein the probe selectively labels a phosphorylated conformation of Btk. In another embodiment is a Btk affinity probe wherein the phosphorylated conformation of Btk is either an active or inactive form of Btk. In a further embodiment is a Btk affinity probe wherein the phosphorylated conformation of Btk is an active form of Btk. In one embodiment is a Btk affinity probe wherein the probe is cell permeable.

In one aspect is a method for assessing the efficacy of a potential Btk inhibitor in a mammal， comprising administering a potential Btk inhibitor to the mammal， administering the Btk affinity probe described herein to the mammal or to cells isolated from the mammal； measuring the activity of the reporter moiety of the Btk affinity probe， and comparing the activity of the reporter moiety to a standard.

In another aspect is a method for assessing the pharmacodynamics of a Btk inhibitor in a mammal， comprising administering a Btk inhibitor to the mammal， administering the Btk affinity probe presented herein to the mammal or to cells isolated from the mammal， and measuring the activity of the reporter moiety of the Btk affinity probe at different time points following the administration of the inhibitor.

In a further aspect is a method for in vitro labeling of a Btk enzyme comprising contacting an active Btk enzyme with the Btk affinity probe described herein. In one embodiment is a method for in vitro labeling of a Btk enzyme wherein the contacting step comprises incubating the active Btk enzyme with the Btk affinity probe presented herein.

In another aspect is a method for in vitro labeling of a Btk enzyme comprising contacting cells or tissues expressing the Btk enzyme with an Btk affinity probe described herein.

In one aspect is a method for detecting a labeled Btk enzyme comprising separating proteins， the proteins comprising a Btk enzyme labeled by an Btk affinity probe described herein， by electrophoresis and detecting the Btk affinity probe by fluorescence.

The present invention includes the following technical solutions：

Technical Solution 1

A compound having a general formula (Ia) or (Ib) or (Ib-l) or (Id) ， or a pharmaceutical acceptable salt thereof：

wherein R is selected from the group consisting of a bond， carbonylalkyleneamino， ( ( (azacycloalk-2-yl) -alkyl) oxomethane) -1， N-diyl； preferably R is selected from the group consisting of ( ( (C_5-7azacycloalk-2-yl) -C_0-2alkyl) oxomethane) -1， N-diyl) ；

wherein R₁ is selected from the group consisting of alkyl， arylalkyl， hydroxyalkyl， aminocarbonylalkyl， carboxylalkyl， aminoalkyl and heteroarylalkyl； preferably R₁ is selected from the group consisting of arylalkyl， hydroxyalkyl， aminocarbonylalkyl， carboxylalkyl， aminoalkyl and heteroarylalkyl； or preferably R₁ is selected from the group consisting of

and

or preferably R₁ is selected from the group consisting of

and

wherein

p is 0， 1， 2 or 3， preferably 0；

q is 0， 1， 2 or 3， preferably 0；

and

Technical Solution 2

A compound or a pharmaceutical acceptable salt thereof， wherein said compound is selected from the group consisting of：

Technical Solution 3

A pharmaceutical composition containing a therapeutically effective amount of a compound according to any of Technical Solutions 1-2 or a pharmaceutical acceptable salt thereof， in combination with one or more pharmaceutically acceptable carriers.

Technical Solution 4

Use of a compound according to any of Technical Solutions 1-2 or a pharmaceutical acceptable salt thereof in manufacture of a medicament for treating or preventing a disease selected from the group consisting of autoimmune disease，

disease， inflammatory disease， cancer， and thromboembolic disease.

Technical Solution 5

Use of a compound according to

Technical Solution

1 or 2 in the preparation of a Btk affinity probe.

Technical Solution 6

Use of any of the following compounds in the preparation of a Btk affinity probe：

Technical Solution 7

A Btk affinity probe as represented by the following formula (Ic) ：

wherein the Btk inhibitor moiety is derivable from a compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) ：

and

or preferably R₁ is selected from the group consisting of

and

wherein

p is 0， 1， 2 or 3， preferably 0；

q is 0， 1， 2 or 3， preferably 0；

R₁₁ is a substituent containing an end group selected from OH， COOH， CONH₂， NH₂， and a nitrogen containing heterocycle (for example， the derivation can be made by removing a hydrogen atom from the end group of R₁₁) ； preferably R₁₁ is an alkyl group substituted by a substituent selected from the group consisting of OH， COOH， CONH₂， NH₂， and a nitrogen containing heterocycle as end group， wherein one or more CH₂ moieties in said alkyl group are optionally replaced with a divalent group selected from the group consisting of -NH-， -CO-， -SO₂-and -SO-； more preferably， R₁₁ is selected from the group consisting of

and

wherein

forms a N-containing heterocycle；

R^a is hydrogen or alkyl；

wherein the linker moiety is selected from a bond， an optionally substituted alkyl moiety， an optionally substituted heterocycle moiety， an optionally substituted amide moiety， a ketone moiety， an optionally substituted carbamate moiety， an ester moiety， or a combination thereof；

wherein the reporter moiety is selected from the group consisting of a label， a dye， a photocrosslinker， a cytotoxic compound， a drug， an affinity label， a photoaffinity label， a reactive compound， an antibody or antibody fragment， a biomaterial， a nanoparticle， a spin label， a fluorophore， a metal-containing moiety， a radioactive moiety， a novel functional group， a group that covalently or noncovalently interacts with other molecules， a photocaged moiety， an actinic radiation excitable moiety， a ligand， a photoisomerizable moiety， biotin， a biotin analogue， a moiety incorporating a heavy atom， a chemically cleavable group， a photocleavable group， a redox-active agent， an isotopically labeled moiety， a biophysical probe， a phosphorescent group， a chemiluminescent group， an electron dense group， a magnetic group， an intercalating group， a chromophore， an energy transfer agent， a biologically active agent， a detectable label， or a combination thereof.

Technical Solution 8

The Btk affinity probe according to Technical Solution 7， wherein the linker moiety is a bond.

Technical Solution 9

The Btk affinity probe according to Technical Solution 7， wherein the reporter moiety is a fluorophore.

Technical Solution 10

The Btk affinity probe according to Technical Solution 9， wherein the fluorophore is a Bodipy fluorophore.

Technical Solution 11

The Btk affinity probe according to Technical Solution 10， wherein the Bodipy fluorophore is a Bodipy FL fluorophore.

Technical Solution 12

A Btk affinity probe having a structure of

Technical Solution 13

A method for assessing the efficacy of a potential Btk inhibitor in a mammal， comprising administering a potential Btk inhibitor to the mammal， administering the Btk affinity probe of any of Technical Solutions 7-12 to the mammal or to cells isolated from the mammal； measuring the activity of the reporter moiety of the Btk affinity probe， and comparing the activity of the reporter moiety to a standard.

Technical Solution 14

A method for assessing the pharmacodynamics of a Btk inhibitor in a mammal， comprising administering a Btk inhibitor to a plurality of mammals， administering the Btk affinity probe of Technical Solutions 7-12 to the plurality of mammals or to cells isolated from a plurality of mammals， and measuring the activity of the reporter moiety of the Btk affinity probe at different time points following the administration of the inhibitor.

Technical Solution 15

A method for in vitro labeling of a Btk enzyme comprising contacting cells or tissues expressing the Btk enzyme with the Btk affinity probe of Technical Solutions 7-12.

Technical Solution 16

A method for detecting a labeled Btk enzyme comprising separating proteins， the proteins comprising a Btk enzyme labeled by the Btk affinity probe of Technical Solutions 7-12， by electrophoresis and detecting the Btk affinity probe by fluorescence.

It is to be understood that the methods and compositions described herein are not limited to the particular methodology， protocols， cell lines， constructs， and reagents described herein and in some embodiments will vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only， and is not intended to limit the scope of the methods and compositions described herein， which will be limited only by the appended claims.

As used herein and in the appended claims， the singular forms “a， ” “an， ” and “the” include plural reference unless the context clearly indicates otherwise.

All publications， patent applications， and patents mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing， for example， the constructs and methodologies that are described in the publications.

The term “alkyl， ” by itself or as part of another molecule means， unless otherwise stated， a straight or branched chain， or cyclic hydrocarbon radical， or combination thereof. In some embodiments， the alkyl chain is fully saturated， mono-or polyunsaturated. In other embodiments， the alkyl chain includes di-and multivalent radicals， having the number of carbon atoms designated (i.e. C₀-C₁₀ or C₀-₁₀ means zero to ten carbons， and C₀-alkyl means a bond) . In other embodiments， examples of saturated hydrocarbon radicals include， but are not limited to， groups such as methyl， ethyl， n-propyl， isopropyl， n-butyl， t-butyl， isobutyl， sec-butyl， cyclohexyl， (cyclohexyl) methyl， cyclopropylmethyl， homologs and isomers of， for example， n-pentyl， n-hexyl， n-heptyl， n-octyl， and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. In a further embodiment， examples of unsaturated alkyl groups include， but are not limited to， vinyl， 2-propenyl， crotyl， 2-isopentenyl， 2- (butadienyl) ， 2， 4-pentadienyl， 3- (1， 4-pentadienyl) ， ethynyl， 1-and 3-propynyl， 3-butynyl， and the higher homologs and isomers. In yet further embodiments， the term “alkyl， ” unless otherwise noted， includes those derivatives of alkyl defined in more detail herein， such as “heteroalkyl” ， “haloalkyl” and “homoalkyl” .

The term “biophysical probe” as used herein refers to probes which detect or monitor structural changes in molecules. In some embodiments， such molecules include， but are not limited to， proteins and the “biophysical probe” is used to detect or monitor interaction of proteins with other macromolecules. In other embodiments， examples of biophysical probes include， but are not limited to， spin-labels， fluorophores， and photoactivatable groups.

The term “carbonyl” as used herein refers to a group containing a moiety selected from the group consisting of-C (O) -， -S (O) -， -S (O) ₂-， and -C (S) -， including， but not limited to， groups containing a least one ketone group， and/or at least one aldehyde group， and/or at least one ester group， and/or at least one carboxylic acid group， and/or at least one thioester group. Such carbonyl groups include ketones， aldehydes， carboxylic acids， esters， and thioesters. In some embodiments， such groups are a part of linear， branched， or cyclic molecules.

The term “chemiluminescent group” as used herein refers to a group which emits light as a result of a chemical reaction without the addition of heat. By way of example only， luminol (5-amino-2， 3-dihydro-1， 4-phthalazinedione) reacts with oxidants like hydrogen peroxide (H₂O₂) in the presence of a base and a metal catalyst to produce an excited state product (3-aminophthalate， 3-APA) .

The term “chromophore” as used herein refers to a molecule which absorbs light of visible wavelengths， UV wavelengths or IR wavelengths.

The term “Cys 481” as used herein refers to the cysteine found in kinases in Fig. 5 at the position corresponding to Cys 481 in Btk (i.e.， the “C” highlighted in bold) .

In other embodiments， the term “detectable label” as used herein refers to a label which is observable using analytical techniques including， but not limited to， fluorescence， chemiluminescence， electron-spin resonance， ultraviolet/visible absorbance spectroscopy， mass spectrometry， nuclear magnetic resonance， magnetic resonance， and electrochemical methods.

The term “dye” as used herein refers to a soluble， coloring substance which contains a chromophore.

The term “electron dense group” as used herein refers to a group which scatters electrons when irradiated with an electron beam. Such groups include， but are not limited to， ammonium molybdate， bismuth subnitrate cadmium iodide， 99％， carbohydrazide， ferric chloride hexahydrate， hexamethylene tetramine， 98.5％， indium trichloride anhydrous， lanthanum nitrate， lead acetate trihydrate， lead citrate trihydrate， lead nitrate， periodic acid， phosphomolybdic acid， phosphotungstic acid， potassium ferricyanide， potassium ferrocyanide， ruthenium red， silver nitrate， silver proteinate (Ag Assay： 8.0-8.5％) “Strong” ， silver tetraphenylporphin (S-TPPS) ， sodium chloroaurate， sodium tungstate， thallium nitrate， thiosemicarbazide (TSC) ， uranyl acetate， uranyl nitrate， and vanadyl sulfate.

In other embodiments， the term “energy transfer agent” as used herein refers to a molecule which either donates or accepts energy from another molecule. By way of example only， fluorescence resonance energy transfer (FRET) is a dipole-dipole coupling process by which the excited-state energy of a fluorescence donor molecule is non-radiatively transferred to an unexcited acceptor molecule which then fluorescently emits the donated energy at a longer wavelength.

The terms “enhance” or “enhancing” means to increase or prolong either in potency or duration a desired effect. By way of example， “enhancing” the effect of therapeutic agents refers to the ability to increase or prolong， either in potency or duration， the effect of therapeutic agents on during treatment of a disease， disorder or condition. An “enhancing-effective amount” as used herein refers to an amount adequate to enhance the effect of a therapeutic agent in the treatment of a disease， disorder or condition. When used in a patient， amounts effective for this use will depend on the severity and course of the disease， disorder or condition， previous therapy， the patient′s health status and response to the drugs， and the judgment of the treating physician.

The term “fluorophore” as used herein refers to a molecule which upon excitation emits photons and is thereby fluorescent.

In some embodiments， the term “label” as used herein refers to a substance which is incorporated into a compound and is readily detected， whereby its physical distribution is detected and/or monitored.

The term “linkage” as used herein to refer to bonds or a chemical moiety formed from a chemical reaction between the functional group of a linker and another molecule. In some embodiments， such bonds include， but are not limited to， covalent linkages and non-covalent bonds， while such chemical moieties include， but are not limited to， esters， carbonates， imines， phosphate esters， hydrazones， acetals， orthoesters， peptide linkages， and oligonucleotide linkages. Hydrolytically stable linkages means that the linkages are substantially stable in water and do not react with water at useful pH values， including but not limited to， under physiological conditions for an extended period of time， perhaps even indefinitely. Hydrolytically unstable or degradable linkages means that the linkages are degradable in water or in aqueous solutions， including for example， blood. In other embodiments， enzymatically unstable or degradable linkages means that the linkage is degraded by one or more enzymes. By way of example only， PEG and related polymers include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. Such degradable linkages include， but are not limited to， ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent， wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include but are not limited to carbonate linkages； imine linkages resulted from reaction of an amine and an aldehyde； phosphate ester linkages formed by reacting an alcohol with a phosphate group； hydrazone linkages which are reaction product of a hydrazide and an aldehyde； acetal linkages that are the reaction product of an aldehyde and an alcohol； orthoester linkages that are the reaction product of a formate and an alcohol； peptide linkages formed by an amine group， including but not limited to， at an end of a polymer such as PEG， and a carboxyl group of a peptide； and oligonucleotide linkages formed by a phosphoramidite group， including but not limited to， at the end of a polymer， and a 5′hydroxyl group of an oligonucleotide.

The phrase “measuring the activity of the reporter moiety” (or a similarly worded phrase) refers to methods for quantifying (in absolute， approximate or relative terms) the reporter moiety in a system under study. In some embodiments， such methods include any methods that quantify a reporter moiety that is a dye； a photocrosslinker； a cytotoxic compound； a drug； an affinity label； a photoaffinity label； a reactive compound； an antibody or antibody fragment； a biomaterial； a nanoparticle； a spin label； a fluorophore， a metal-containing moiety； a radioactive moiety； a novel functional group； a group that covalently or noncovalently interacts with other molecules； a photocaged moiety； an actinic radiation excitable moiety； a ligand； a photoisomerizable moiety； biotin； a biotin analogue； a moiety incorporating a heavy atom； a chemically cleavable group； a photocleavable group； a redox-active agent； an isotopically labeled moiety； a biophysical probe； a phosphorescent group； a chemiluminescent group； an electron dense group； a magnetic group； an intercalating group； a chromophore； an energy transfer agent； a biologically active agent； a detectable label； and any combination of the above.

The term “moiety incorporating a heavy atom” as used herein refers to a group which incorporates an ion of atom which is usually heavier than carbon. In some embodiments， such ions or atoms include， but are not limited to， silicon， tungsten， gold， lead， and uranium.

The term “nanoparticle” as used herein refers to a particle which has a particle size between about 500 nm to about 1 nm.

The term “pharmaceutically acceptable” as used herein refers to a material， including but not limited， to a salt， carrier or diluent， which does not abrogate the biological activity or properties of the compound， and is relatively nontoxic. In one embodiment， the material is administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The term “photoaffinity label” as used herein refers to a label with a group， which， upon exposure to light， forms a linkage with a molecule for which the label has an affinity. By way of example only， in some embodiments， such a linkage is covalent or non-covalent.

The term “photocaged moiety” as used herein refers to a group which， upon illumination at certain wavelengths， covalently or non-covalently binds other ions or molecules.

The term “photoisomerizable moiety” as used herein refers to a group wherein upon illumination with light changes from one isomeric form to another.

The term “radioactive moiety” as used herein refers to a group whose nuclei spontaneously give off nuclear radiation， such as alpha， beta， or gamma particles； wherein， alpha particles are helium nuclei， beta particles are electrons， and gamma particles are high energy photons.

The term “spin label” as used herein refers to molecules which contain an atom or a group of atoms exhibiting an unpaired electron spin (i.e. a stable paramagnetic group) that in some embodiments are detected by electron spin resonance spectroscopy and in other embodiments are attached to another molecule. Such spin-label molecules include， but are not limited to， nitryl radicals and nitroxides， and in some embodiments are single spin-labels or double spin-labels.

The phrase ″therapeutically effective amount″ of the compound of the invention means a sufficient amount of the compound to treat disorders， at a reasonable benefit/risk ratio applicable to any medical treatment. It is understood， however， that the total daily usage of the compounds and compositions of the invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient depends upon a variety of factors including the disorder being treated and the severity of the disorder； activity of the specific compound employed； the specific composition employed； the age， body weight， general health， sex and diet of the patient； the time of administration， route of administration， and rate of excretion of the specific compound employed； the duration of the treatment； drugs used in combination or coincidental with the specific compound employed； and like factors well-known in the medical arts. For example， it is well within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

The term ″pharmaceutically acceptable carrier″ as used herein， means a non-toxic， inert solid， semi-solid or liquid filler， diluent， encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as， but not limited to， lactose， glucose and sucrose； starches such as， but not limited to， corn starch and potato starch； cellulose and its derivatives such as， but not limited to， sodium carboxymethyl cellulose， ethyl cellulose and cellulose acetate； powdered tragacanth； malt； gelatin； talc； excipients such as， but not limited to， cocoa butter and suppository waxes； oils such as， but not limited to， peanut oil， cottonseed oil， safflower oil， sesame oil， olive oil， corn oil and soybean oil； glycols； such a propylene glycol； esters such as， but not limited to， ethyl oleate and ethyl laurate； agar； buffering agents such as， but not limited to， magnesium hydroxide and aluminum hydroxide； alginic acid； pyrogen-free water； isotonic saline； Ringer′s solution； ethyl alcohol， and phosphate buffer solutions， as well as other non-toxic compatible lubricants such as， but not limited to， sodium lauryl sulfate and magnesium stearate， as well as coloring agents， releasing agents， coating agents， sweetening， flavoring and perfuming agents， preservatives and antioxidants can also be present in the composition， according to the judgment of the formulator.

The term “subject” as used herein， refers to an animal which is the object of treatment， observation or experiment. In one embodiment the subject is a mammal including， but not limited to， a human.

In some embodiments， the term “substituents” also referred to as “non-interfering substituents” “refers to groups which are used to replace another group on a molecule. Such groups include， but are not limited to， halo， C₁-C₁₀alkyl， C₂-C₁₀alkenyl， C₂-C₁₀alkynyl， C₁-C₁₀alkoxy， C₅-C₁₂aralkyl， C₃-C₁₂cycloalkyl， C₄-C₁₂cycloalkenyl， phenyl， substituted phenyl， toluolyl， xylenyl， biphenyl， C₂-C₁₂alkoxyalkyl， C₅-C₁₂alkoxyaryl， C₅-C₁₂aryloxyalkyl， C₇-C₁₂oxyaryl， C₁-C₆alkylsulfinyl， C₁-C₁₀alkylsulfonyl， - (CH₂) _m-O- (C₁-C₁₀alkyl) wherein m is from 1 to 8， aryl， substituted aryl， substituted alkoxy， fluoroalkyl， heterocyclic radical， substituted heterocyclic radical， nitroalkyl， -NO₂， -CN， -NRC (O) - (C₁-C₁₀alkyl) ， -C(O) - (C₁-C₁₀alkyl) ， C₂-C₁₀alkthioalkyl， -C (O) O- (C₁-C₁₀alkyl) ， -OH， -SO₂，＝S， -COOH， -NR₂， carbonyl， -C (O) - (C₁-C₁₀alkyl) -CF₃， -C (O) -CF₃， -C (O) NR₂， - (C ₁-C₁₀aryl) -S- (C₆-C₁₀aryl) ， -C (O) - (C₆-C₁₀aryl) ， - (CH₂) m-O- (CH₂) m-O- (C₁-C₁₀alkyl) wherein each m is from 1 to 8， -C (O) NR₂， -C (S) NR₂， -SO₂NR₂， -NRC (O) NR₂， -NRC (S) NR₂， salts thereof， and the like. In some embodiments， each R group in the preceding list includes， but is not limited to， H， alkyl or substituted alkyl， aryl or substituted aryl， or alkaryl. Where substituent groups are specified by their conventional chemical formulas， written from left to right， they equally encompass the chemically identical substituents that would result from writing the structure from right to left； for example， -CH₂O-is equivalent to -OCH₂-.

In some embodiments， and by way of example only， substituents for alkyl and heteroalkyl radicals (including those groups referred to as alkylene， alkenyl， heteroalkylene， heteroalkenyl， alkynyl， cycloalkyl， heterocycloalkyl， cycloalkenyl， and heterocycloalkenyl) includes， but is not limited to： -OR，＝O，＝NR，＝N-OR， -NR₂， -SR， -halogen， -SiR₃， -OC (O) R， -C (O) R， -CO₂R， -CONR₂， -OC (O) NR₂， -NRC (O) R， -NRC (O) NR₂， -NR (O) ₂R， -NR-C (NR₂) ＝NR， -S (O) R， -S (O) ₂R， -S (O) ₂NR₂， -NRSO₂R， -CN and -NO₂. In further embodiments， each R group in the preceding list includes， but is not limited to， hydrogen， substituted or unsubstituted heteroalkyl， substituted or unsubstituted aryl， including but not limited to， aryl substituted with 1-3 halogens， substituted or unsubstituted alkyl， alkoxy or thioalkoxy groups， or aralkyl groups. In some embodiments when two R groups are attached to the same nitrogen atom， they are combined with the nitrogen atom to form a 5-， 6-， or 7-membered ring. In other embodiments for example， -NR₂ includes， but is not be limited to， 1-pyrrolidinyl and 4-morpholinyl.

In other embodiments and by way of example， substituents for aryl and heteroaryl groups include， but are not limited to， -OR，＝O，＝NR，＝N-OR， -NR₂， -SR， -halogen， -SiR3， -OC (O) R， -C (O) R， -CO₂R， -CONR₂， -OC (O) NR₂， -NRC (O) R， -NRC (O) NR₂， -NR(O) ₂R， -NR-C (NR₂) ＝NR， -S (O) R， -S (O) ₂R， -S (O) ₂NR2， -NRSO₂R， -CN， -NO₂， -R， -N₃， -CH (Ph) ₂， fluoro (C₁-C₄) alkoxy， and fluoro (C₁-C₄) alkyl， in a number ranging from zero to the total number of open valences on the aromatic ring system. In a further embodiment， each R group in the preceding list includes， but is not limited to， hydrogen， alkyl， heteroalkyl， aryl and heteroaryl.

Unless otherwise indicated， conventional methods of mass spectroscopy， NMR， HPLC， protein chemistry， biochemistry， recombinant DNA techniques and pharmacology， are employed.

Compounds presented herein include isotopically-labeled compounds， which are identical to those recited in the various formulas and structures presented herein， but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. In some embodiments examples of isotopes that are incorporated into the present compounds include isotopes of hydrogen， carbon， nitrogen， oxygen， fluorine and chlorine， such as ²H， ³H， ¹³C， ¹⁴C， ¹⁵N， ¹⁸O， ¹⁷O， ³⁵S， ¹⁸F， ³⁶C1， respectively. Certain isotopically-labeled compounds described herein， for example those into which radioactive isotopes such as ³H and ¹⁴c are incorporated， are useful in drug and/or substrate tissue distribution assays. Further， in other embodiments， substitution with isotopes such as deuterium， i.e.， ²H， affords certain therapeutic advantages resulting from greater metabolic stability， for example increased in vivo half-life or reduced dosage requirements.

In other embodiments， are compounds described herein having asymmetric carbon atoms and therefore exist as enantiomers or diastereomers. In some embodiments， diastereomeric mixtures are separated into their individual diastereomers on the basis of their physical chemical differences， for example， by chromatography and/or fractional crystallization. In other embodiments enantiomers are separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g.， alcohol) ， separating the diastereomers and converting (e.g.， hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers， including diastereomers， enantiomers， and mixtures thereof are considered as part of the compositions described herein.

In additional or further embodiments， the compounds described herein are used in the form of pro-drugs. In additional or further embodiments， the compounds described herein are metabolized upon administration to an organism in need to produce a metabolite that is then used to produce a desired effect， including a desired therapeutic effect.

The methods and formulations described herein include the use of N-oxides， crystalline forms (also known as polymorphs) ， or pharmaceutically acceptable salts. In certain embodiments， compounds described herein exist as tautomers. All tautomers are included within the scope of the compounds presented herein. In some embodiments， the compounds described herein exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water， ethanol， and the like. The solvated forms of the compounds presented herein are also considered to be disclosed herein.

In further embodiments are compounds described herein which exist in several tautomeric forms. All such tautomeric forms are considered as part of the compositions described herein. Also， for example in some embodiments， all enol-keto forms of any compounds herein are considered as part of the compositions described herein.

In other embodiments， are compounds described herein are acidic and in some embodiments form a salt with a pharmaceutically acceptable cation. Some of the compounds herein are basic and in some embodiments， form a salt with a pharmaceutically acceptable anion. All such salts， including di-salts are within the scope of the compositions described herein and in some embodiments are prepared by conventional methods. For example， in other embodiments salts are prepared by contacting the acidic and basic entities， in either an aqueous， non-aqueous or partially aqueous medium. The salts are recovered by using at least one of the following techniques： filtration， precipitation with a non-solvent followed by filtration， evaporation of the solvent， or， in the case of aqueous solutions， lyophilization.

In some embodiments， pharmaceutically acceptable salts of the compounds disclosed herein are formed when an acidic proton present in the parent compound either is replaced by a metal ion， by way of example an alkali metal ion， an alkaline earth ion， or an aluminum ion； or coordinates with an organic base. In addition， in other embodiments， the salt forms of the disclosed compounds are prepared using salts of the starting materials or intermediates.

In further embodiments， the type of pharmaceutical acceptable salts， include， but are not limited to： (1) acid addition salts， formed with inorganic acids such as hydrochloric acid， hydrobromic acid， sulfuric acid， nitric acid， phosphoric acid， and the like； or formed with organic acids such as acetic acid， propionic acid， hexanoic acid， cyclopentanepropionic acid， glycolic acid， pyruvic acid， lactic acid， malonic acid， succinic acid， malic acid， maleic acid， fumaric acid， tartaric acid， citric acid， benzoic acid， 3- (4-hydroxybenzoyl) benzoic acid， cinnamic acid， mandelic acid， methanesulfonic acid， ethanesulfonic acid， 1， 2-ethanedisulfonic acid， 2-hydroxyethanesulfonic acid， benzenesulfonic acid， 2-naphthalenesulfonic acid， 4-methylbicyclo- [2.2.2] oct-2-ene-1-carboxylic acid， glucoheptonic acid， 4， 4′-methylenebis- (3-hydroxy-2-ene-l-carboxylic acid) ， 3-phenylpropionic acid， trimethylacetic acid， tertiary butylacetic acid， lauryl sulfuric acid， gluconic acid， glutamic acid， hydroxynaphthoic acid， salicylic acid， stearic acid， muconic acid， and the like； (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion， e.g.， an alkali metal ion， an alkaline earth ion， or an aluminum ion； or coordinates with an organic base. In yet a further embodiment， acceptable organic bases include ethanolamine， diethanolamine， triethanolamine， tromethamine， N-methylglucamine， and the like. In some embodiments， acceptable inorganic bases include aluminum hydroxide， calcium hydroxide， potassium hydroxide， sodium carbonate， sodium hydroxide， and the like.

In some embodiments， the corresponding counter-ions of the pharmaceutically acceptable salts are analyzed and identified using various methods including， but not limited to， ion exchange chromatography， ion chromatography， capillary electrophoresis， inductively coupled plasma， atomic absorption spectroscopy， mass spectrometry， or any combination thereof.

It should be understood that in some embodiments， a reference to a salt includes the solvent addition forms or crystal forms thereof， particularly solvates or polymorphs. In further embodiments， solvates contain either stoichiometric or non-stoichiometric amounts of a solvent， and are often formed during the process of crystallization with pharmaceutically acceptable solvents such as water， ethanol， and the like. In yet another embodiment， hydrates are formed when the solvent is water， or alcoholates are formed when the solvent is alcohol. Polymorphs include the different crystal packing arrangements of the same elemental composition of a compound. In one embodiment， are polymorphs having different X-ray diffraction patterns， infrared spectra， melting points， density， hardness， crystal shape， optical and electrical properties， stability， and solubility. In further embodiments， various factors such as the recrystallization solvent， rate of crystallization， and storage temperature cause a single crystal form to dominate.

In other embodiments， the screening and characterization of the pharmaceutically acceptable salts， polymorphs and/or solvates is accomplished using a variety of techniques including， but not limited to， thermal analysis， x-ray diffraction， spectroscopy， vapor sorption， and microscopy. In yet a further embodiment， thermal analysis methods address thermo chemical degradation or thermo physical processes including， but not limited to， polymorphic transitions， and such methods are used to analyze the relationships between polymorphic forms， determine weight loss， to find the glass transition temperature， or for excipient compatibility studies. Such methods include， but are not limited to， Differential scanning calorimetry (DSC) ， Modulated Differential Scanning Calorimetry (MDCS) ， Thermogravimetric analysis (TGA) ， and Thermogravi-metric and Infrared analysis (TG/IR) . X-ray diffraction methods include， but are not limited to， single crystal and powder diffractometers and synchrotron sources. The various spectroscopic techniques used include， but are not limited to， Raman， FTIR， UVIS， and NMR (liquid and solid state) . The various microscopy techniques include， but are not limited to， polarized light microscopy， Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX) ， Environmental Scanning Electron Microscopy with EDX (in gas or water vapor atmosphere) ， IR microscopy， and Raman microscopy.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the present methods and compositions are obtained by reference to the following detailed description that sets forth illustrative embodiments， in which the principles of our methods， compositions， devices and apparatuses are utilized， and the accompanying drawings of which：

Fig 1 shows structures of representative Btk inhibitors.

Fig 2 shows structures of representative fluorescent probes.

Fig. 3 shows components of affinity probes；

Fig. 4 shows general scheme of measuring target engagement by competition assays between inhibitors and affinity probes.

Fig. 5 shows sequence alignments of the ATP binding pocket in kinases containing the conserved cysteine (arrow) corresponding to Cys 481 in Btk. The protein accession numbers are also shown.

Fig. 6 shows concentration-dependent labeling of recombinant Btk by probe 14.

Fig. 7 shows time-dependent labeling of recombinant Btk by probe 14.

Fig. 8 shows probe 14 predominantly labeled endogenous Btk in live cells (concentration course) ；

Fig. 9 shows the result of time course experiments in cellular labeling.

Fig. 10 shows the result of immunoprecipitation of Btk from probe 14-labeled lysates. lane 1： cell lysates； lane 2： supernatant after removal of intrinsic IgG； lane 3： supernatant after immunoprecipitation； lane 4： supernatant of the last wash before elution； lane 5： supernatant of the first elution by applying LDS sample buffer onto protein A Sepharose beads.

Fig. 11 shows labeling of Btk by probe 14 (0.5 uM) is completely competed off by ibrutinib and compound 2 (1 uM) .

Fig. 12 shows the measurement of the extent of Btk occupancy by inhibitors (ibrutinib and compound 2) in live cells. Band densitometry is measured by Gelpro32， and Graphpad Prism is used to determine the IC50 values.

Fig. 13 shows the competition experiment in OCI-Ly7 cells (a) and Jurkat cells (b) . 1.5×10⁶ cells are pre-incubated with compounds for 1h at 1μM before labeling with probe 14 for 2h at 0.5μM， then lysed， quantified and analyzed by SDS/PAGE and fluorescent gel scanning (fluorescence， CY2) .

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is the synthesis and characterization of Btk inhibitors. Disclosed herein is also the synthesis and characterization of cell permeable probes that label Btk at a unique， non-catalytic cysteine residue in the ATP binding pocket. Other embodiments disclosed herein demonstrate the utility of such probes in assessing pharmacodynamics in mammals treated with small molecule Btk inhibitors.

Btk Inhibitors

The Btk inhibitor described herein has a general formula (Ia) or (Ib) or (Ib-1) or (Id) ， or a pharmaceutically acceptable salt thereof：

and

and

more preferably， R₁ is selected from the group consisting of

and

wherein

p is 0， 1， 2 or 3， preferably 0；

q is 0， 1， 2 or 3， preferably 0；

and

Examples of the Btk inhibitors according to the present invention include， but are not limited to：

Btk Activity Probe Compounds

The Btk affinity probe compounds described herein are composed of a moiety comprising an inhibitor of Btk (or a Btk inhibitor moiety) ， a linker moiety， and a reporter moiety. In one embodiment， the inhibitor of Btk is an irreversible inhibitor. In another embodiment， the irreversible inhibitor of Btk binds to a non-catalytic residue in the ATP binding pocket of Btk； in further embodiments， the non-catalytic residue is a cysteine residue. In some embodiments， the Btk affinity probe forms a covalent bond with at least one non-catalytic residue of Btk. In other embodiments， the Btk affinity probe forms a non-covalent bond with at least one non-catalytic residue of Btk. In a further embodiment， the Btk affinity probe forms hydrogen bonding within the ATP binding pocket of Btk. In yet a further embodiment， the Btk affinity probe has Van der Waals attractions with the Btk enzyme.

In some other embodiments， the Btk affinity probes described herein are activity dependent such that the probe binds only an active Btk enzyme. In further embodiments， the Btk affinity probe binds a Btk enzyme that has been switched on by phosphorylation by upstream kinases. In yet a further embodiment， the Btk affinity probes described herein are activity independent such that the probe binds Btk enzymes that have not been switched on by phosphorylation by upstream kinases. In some embodiments， the Btk affinity probe labels a phosphorylated conformation of a Btk enzyme. In other embodiments， the Btk affinity probe labels a Btk in a non-phosphorylated conformation.

In some embodiments， the Btk affinity probe is permeable to cells.

In further embodiments， the linker moiety is selected from a bond， a substituted alkyl moiety， a substituted heterocycle moiety， a substituted amide moiety， a ketone moiety， a substituted carbamate moiety， an ester moiety， or any combination thereof. In further embodiments， the reporter moiety is a moiety that is detected using standard or modified laboratory equipment.

wherein the Btk inhibitor moiety is derivable from the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) ；

wherein

forms a N-containing heterocycle；

R^a is hydrogen or alkyl.

In one embodiment， the Btk inhibitor moiety is derived from a Btk inhibitor having a general formula (Ia) or (Ib) or (Ib-1) or (Id) ：

wherein R is selected from the group consisting of a bond， carbonylalkyleneamino (e.g.， carbonylC_1-6alkyleneamino) ， ( ( (azacycloalk-2-yl) -alkyl) oxomethane) -1， N-diyl (e.g. ( ( (C_5-7azacycloalk-2-yl) -C_0-2alkyl) oxomethane) -1， N-diyl) ； preferably， R is selected from the group consisting of

and

and

more preferably， R₁ is selected from the group consisting of

and

wherein

p is 0， 1， 2 or 3， preferably 0；

q is 0， 1， 2 or 3， preferably 0；

and

Examples of the Btk inhibitors from which the Btk inhibitor moiety according to the present invention is derived， include， but are not limited to：

In one embodiment， the Btk inhibitor moiety is selected from the group consisting of

In one embodiment， the Btk inhibitor moiety is derived from an irreversible inhibitor of Btk. In some embodiments， such irreversible inhibitors of Btk should possess at least one of the following characteristics： potency， selectively and cell permeability. In further embodiments， such irreversible inhibitors of Btk possess at least two of the aforementioned characteristics， and in further embodiments， at least all of the aforementioned characteristics.

In another embodiment， the linker moiety is selected from a bond， a polymer， a water soluble polymer， optionally substituted alkyl， optionally substituted heteroalkyl， optionally substituted heterocycloalkyl， optionally substituted cycloalkyl， optionally substituted heterocycloalkylalkyl， optionally substituted heterocycloalkylalkenyl， optionally substituted aryl， optionally substituted heteroaryl， and optionally substituted heterocycloalkylalkenylalkyl. In some embodiments， the linker moiety is selected from a bond. In some embodiments， the linker moiety is an optionally substituted heterocycle. In other embodiments， the heterocycle is selected from aziridine， oxirane， episulfide， azetidine， oxetane， pyrroline， tetrahydrofuran， tetrahydrothiophene， pyrrolidine， pyrazole， pyrrole， imidazole， triazole， tetrazole， oxazole， isoxazole， oxirene， thiazole， isothiazole， dithiolane， furan， thiophene， piperidine， tetrahydropyran， thiane， pyridine， pyran， thiapyrane， pyridazine， pyrimidine， pyrazine， piperazine， oxazine， thiazine， dithiane， and dioxane. In some embodiments， the heterocycle is piperazine. In further embodiments， the linker moiety is optionally substituted with halogen， CN， OH， NO₂， alkyl， S (O) ， and S (O) ₂. In other embodiments， the water soluble polymer is a PEG group.

In other embodiments， the linker moiety provides sufficient spatial separation between the reporter moiety and the Btk inhibitor moiety. In further embodiments， the linker moiety is stable. In yet a further embodiment， the linker moiety does not substantially affect the response of the reporter moiety. In other embodiments the linker moiety provides chemical stability to the Btk affinity probe. In further embodiments， the linker moiety provides sufficient solubility to the Btk affinity probe.

In some embodiments， linkages such as water soluble polymers are coupled at one end to a Btk inhibitor moiety and to a reporter moiety at the other end. In other embodiments， the water soluble polymers are coupled via a functional group or substituent of the Btk inhibitor moiety. In further embodiments， the water soluble polymers are coupled via a functional group or substituent of the reporter moiety. In other embodiments， covalent attachment of hydrophilic polymers to a Btk inhibitor moiety and a reporter moiety represents one approach to increasing water solubility (such as in a physiological environment) ， bioavailability， increasing serum half-life， increasing pharmacodynamic parameters， or extending the circulation time of the Btk affinity probe， including proteins， peptides， and particularly hydrophobic molecules. In further embodiments， additional important features of such hydrophilic polymers include biocompatibility and lack of toxicity. In other embodiments， for therapeutic use of the end-product preparation， the polymer is pharmaceutically acceptable.

In some embodiments， examples of hydrophilic polymers include， but are not limited to： polyalkyl ethers and alkoxy-capped analogs thereof (e.g.， polyoxyethylene glycol， polyoxyethylene/propylene glycol， and methoxy or ethoxy-capped analogs thereof， polyoxyethylene glycol， the latter is also known as polyethylene glycol or PEG) ； polyvinylpyrrolidones； polyvinylalkyl ethers； polyoxazolines， polyalkyl oxazolines and polyhydroxyalkyl oxazolines； polyacrylamides， polyalkyl acrylamides， and polyhydroxyalkyl acrylamides (e.g.， polyhydroxypropylmethacrylamide and derivatives thereof) ； polyhydroxyalkyl acrylates； polysialic acids and analogs thereof， hydrophilic peptide sequences； polysaccharides and their derivatives， including dextran and dextran derivatives， e.g.， carboxymethyldextran， dextran sulfates， aminodextran； cellulose and its derivatives， e.g.， carboxymethyl cellulose， hydroxyalkyl celluloses； chitin and its derivatives， e.g.， chitosan， succinyl chitosan， carboxymethylchitin， carboxymethylchitosan； hyaluronic acid and its derivatives； starches； alginates； chondroitin sulfate； albumin； pullulan and carboxymethyl pullulan； polyaminoacids and derivatives thereof， e.g.， polyglutamic acids， polylysines， polyaspartic acids， polyaspartamides； maleic anhydride copolymers such as： styrene maleic anhydride copolymer， divinylethyl ether maleic anhydride copolymer； polyvinyl alcohols； copolymers thereof， terpolymers thereof， mixtures thereof， and derivatives of the foregoing. In other embodiments， the water soluble polymer is any structural form including but not limited to linear， forked or branched. In some embodiments， polymer backbones that are water-soluble， with from 2 to about 300 termini， are particularly useful. In further embodiments， multifunctional polymer derivatives include， but are not limited to， linear polymers having two termini， each terminus being bonded to a functional group which is the same or different. In some embodiments， the water polymer comprises a poly (ethylene glycol) moiety. In further embodiments， the molecular weight of the polymer is of a wide range， including but not limited to， between about 100 Da and about 100,000 Da or more. In yet further embodiments， the molecular weight of the polymer is between about 100 Da and about 100,000 Da， including but not limited to， about 100,000 Da， about 95,000 Da， about 90,000 Da， about 85,000 Da， about 80,000 Da， about 75,000 Da， about 70,000 Da，about 65,000 Da， about 60,000 Da， about 55,000 Da， about 50,000 Da， about 45,000 Da， about 40,000 Da， about 35,000 Da， 30,000 Da， about 25,000 Da， about 20,000 Da， about 15,000 Da， about 10,000 Da， about 9,000 Da， about 8,000 Da， about 7,000 Da， about 6,000 Da， about 5,000 Da， about 4,000 Da， about 3,000 Da， about 2,000 Da， about 1,000 Da， about 900 Da， about 800 Da， about 700 Da， about 600 Da， about 500 Da， about 400 Da， about 300 Da， about 200 Da， and about 100 Da. In some embodiments， the molecular weight of the polymer is between about 100 Da and 50,000 Da. In some embodiments， the molecular weight of the polymer is between about 100 Da and 40,000 Da. In some embodiments， the molecular weight of the polymer is between about 1,000 Da and 40,000 Da. In some embodiments， the molecular weight of the polymer is between about 5,000 Da and 40,000 Da. In some embodiments， the molecular weight of the polymer is between about 10,000 Da and 40,000 Da. In some embodiments， the poly (ethylene glycol) molecule is a branched polymer. In further embodiments， the molecular weight of the branched chain PEG is between about 1,000 Da and about 100,000 Da， including but not limited to， about 100,000 Da， about 95,000 Da， about 90,000 Da， about 85,000 Da， about 80,000 Da， about 75,000 Da， about 70,000 Da， about 65,000 Da， about 60,000 Da， about 55,000 Da，about 50,000 Da， about 45,000 Da， about 40,000 Da， about 35,000 Da， about 30,000 Da， about 25,000 Da， about 20,000 Da， about 15,000 Da， about 10,000 Da， about 9,000 Da， about 8,000 Da， about 7,000 Da， about 6,000 Da， about 5,000 Da， about 4,000 Da， about 3,000 Da， about 2,000 Da， and about 1,000 Da. In some embodiments， the molecular weight of the branched chain PEG is between about 1,000 Da and about 50,000 Da. In some embodiments， the molecular weight of the branched chain PEG is between about 1,000 Da and about 40,000 Da. In some embodiments， the molecular weight of the branched chain PEG is between about 5,000 Da and about 40,000 Da. In some embodiments， the molecular weight of the branched chain PEG is between about 5,000 Da and about 20,000 Da. The foregoing list for substantially water soluble backbones is by no means exhaustive and is merely illustrative， and in some embodiments， the polymeric materials having the qualities described above suitable for use in methods and compositions described herein.

In further embodiments， the number of water soluble polymers linked to a Btk inhibitor moiety and a reporter moiety described herein is adjusted to provide an altered (including but not limited to， increased or decreased) pharmacologic， pharmacokinetic or pharmacodynamic characteristic such as in vivo half-life. In some embodiments， the half-life of the Btk affinity probe is increased at least about 10， about 20， about 30， about 40， about 50， about 60， about 70， about 80， about 90 percent， about two fold， about five-fold， about 10-fold， about 50-fold， or at least about 100-fold over a Btk affinity probe without a water soluble linker.

In another embodiment， X is selected from the group consisting of： a bond， -O (CO) -， -NR^a (CO) -， -NR^a-，

-O-， -S-， -S-S-， -O-NR^a-， -O (CO) O-， -O (CO) NR^a， -NR^a (CO) NR^a-， -N＝CR^a-， -S (CO) -， -S (O) -， and-S (O) ₂-；

wherein

forms a N-containing heterocycle. In one embodiment， X is NR^a (CO) . In another embodiment， X is a bond. In another embodiment， X is -O (CO) -.

In a further embodiment， Y is selected from the group consisting of： a bond， -O (CO) -， -NR^a (CO) -， -NR^a-，

wherein

forms a N-containing heterocycle. In yet a further embodiment， Y is a bond. In one embodiment， Y is -NR^a (CO) -. In yet another embodiment， R^a is hydrogen. In yet a further embodiment， R^a is alkyl.

In a further embodiment， the reporter moiety is selected from the group consisting of a label， a dye， a photocrosslinker， a cytotoxic compound， a drug， an affinity label， a photoaffinity label， a reactive compound， an antibody or antibody fragment， a biomaterial， a nanoparticle， a spin label， a fluorophore， a metal-containing moiety， a radioactive moiety， a novel functional group， a group that covalently or noncovalently interacts with other molecules， a photocaged moiety， an actinic radiation excitable moiety， a ligand， a photoisomerizable moiety， biotin， a biotin analog， a moiety incorporating a heavy atom， a chemically cleavable group， a photocleavable group， a redox-active agent， an isotopically labeled moiety， a biophysical probe， a phosphorescent group， a chemiluminescent group， an electron dense group， a magnetic group， an intercalating group， a chromophore， an energy transfer agent， a biologically active agent， a detectable label， or a combination thereof.

In another embodiment， the reporter moiety is a fluorophore. In a further embodiment， the fluorophore is selected from the group consisting of： BODIPY 493/503， BODIPY FL， BODIPY R6G， BODIPY 530/550， BODIPY TMR， BODIPY 558/568， BODIPY 564/570， BODIPY 576/589， BODIPY 581/591， BODIPY TR， Fluorescein， 5 (6) -Carboxyfluorescein， 2， 7-Dichlorofluorescein， N， N-Bis (2， 4， 6-trimethylphenyl) --3， 4： 9， 10-perylenebis (dicarboximide， HPTS， Ethyl Eosin， DY-490XL MegaStokes， DY-485XL MegaStokes， Adirondack Green 520， ATTO 465， ATTO 488， ATTO 495， YOYO-1， 5-FAM， BCECF， BCECF， dichlorofluorescein， rhodamine 110， rhodamine 123， Rhodamine Green， YO-PRO-1， SYTOX Green， Sodium Green， SYBR Green I， Alexa Fluor 500， FITC， Fluo-3， Fluo-4， fluoro-emerald， YoYo-1 ssDNA， YoYo-1 dsDNA， YoYo-1， SYTO RNASelect， Diversa Green-FP， Dragon Green， EvaGreen， Surf Green EX， Spectrum Green， Oregon Green 488， NeuroTrace 500525， NBD-X， MitoTracker Green FM， LysoTracker Green DND-26， CBQCA， PA-GFP (post-activation) ， WEGFP (post-activation) ， FIASH-CCXXCC， Azami Green monomeric， Azami Green， EGFP (Campbell Tsien 2003) ， EGFP (Patterson 2001) ， Fluorescein， Kaede Green， 7-Benzylamino-4-Nitrobenz-2-Oxa-1， 3-Diazole， Bexl， Doxorubicin， Lumio Green， and SuperGlo GFP.

In a further embodiment， the fluorophore is selected from the group consisting of： BODIPY 493/503， BODIPY FL， BODIPY R6G， BODIPY 530/550， BODIPY TMR， BODIPY 558/568， BODIPY 564/570， BODIPY 576/589， BODIPY 581/591， and BODIPY TR. In yet a further embodiment， the fluorophore is BODIPY FL. In certain embodiments， the fluorophore is not BODIPY 530. In some embodiments， the fluorophore has an excitation maxima of between about 500 and about 600 nm. In some other embodiments， the fluorophore has an excitation maxima of between about 500 and about 550 nm. In another embodiments， the fluorophore has an excitation maxima of between about 550 and about 600 nm. In yet a further embodiment， the fluorophore has an excitation maxima of between about 525 and about 575 nm. In other embodiments， the fluorophore has an emission maxima of between about 510 and about 670 nm. In another embodiment， the fluorophore has an emission maxima of between about 510 and about 600 nm. In a further embodiment， the fluorophore has an emission maxima of between about 600 and about 670 nm. In another embodiment， the fluorophore has an emission maxima of between about 575 and about 625 nm.

In some embodiments， the linkage formed is a stable linkage. In other embodiments， in the case where the conjugate comprises two components， the linker moiety forms a linkage， in some embodiments， a stable linkage， between the Btk inhibitor moiety and the reporter moiety. In some embodiments， the linker moiety is stable and provides the means to control and determine the distance between the Btk inhibitor moiety and the report moiety. Further， in some embodiments， the linker moiety is selected such that the probe′s solubility is maintained. In other embodiments， the number and order of units that comprise the linker moiety is selected such that the length between the first component and the second component， as well as the hydrophobic and hydrophilic characteristics of the linker is controlled.

In the present context， spatial separation means a thermochemically and photochemically non-active distance-making group and in some embodiments is used to join two or more different moieties of the types defined above. In other embodiments， spacers are selected on the basis of a variety of characteristics including their hydrophobicity， hydrophilicity， molecular flexibility and length. The spacer， thus， in some embodiments， comprises a chain of carbon atoms optionally interrupted or terminated with one or more heteroatoms， such as oxygen atoms， nitrogen atoms， and/or sulphur atoms. Thus， in some embodiments， the spacer comprises one or more amide， ester， amino， ether， and/or thioether functionalities， and optionally aromatic or mono/polyunsaturated hydrocarbons， polyoxyethylene such as polyethylene glycol， oligo/polyamides such as poly-. α-alanine， polyglycine， polylysine， and peptides in general， oligosaccharides， oligo/polyphosphates. Moreover， in other embodiments， the spacer consists of combined units thereof. In further embodiments， the length of the spacer varies， taking into consideration the desired or necessary positioning and spatial orientation of the active/functional part of the Btk affinity probe.

Without limiting the scope of the compositions described herein， in some embodiments the reporter moiety is Bodipy. In the present context， the term reporter moiety means a group which is detectable either by itself or as a part of a detection series.

In some embodiments， Compound (14) retains the solubility and membrane permeability of Compound (11) ， allowing detection and quantitation of labeled Btk by SDS-PAGE and laser densitometry.

In some embodiments， the labeled Btk affinity probes described herein are purified by one or more procedures including， but are not limited to， affinity chromatography； anion-or cation-exchange chromatography (using， including but not limited to， DEAE SEPHAROSE) ； chromatography on silica； reverse phase HPLC； gel filtration (using， including but not limited to， SEPHADEX G-75) ； hydrophobic interaction chromatography； size-exclusion chromatography， metal-chelate chromatography； ultrafiltration/diafiltration； ethanol precipitation； ammonium sulfate precipitation； chromatofocusing； displacement chromatography； electrophoretic procedures (including but not limited to preparative isoelectric focusing) ， differential solubility (including but not limited to ammonium sulfate precipitation) ， or extraction. In other embodiments， apparent molecular weight is estimated by GPC by comparison to globular protein standards (PROTEIN PURIFICATION METHODS， A PRACTICAL APPROACH (Harris &Angal， Eds. ) IRL Press 1989， 293-306) .

In addition， in some embodiments， the synthetic procedures disclosed below includes various purifications， such as column chromatography， flash chromatography， thin-layer chromatography (TLC) ， recrystallization， distillation， high-pressure liquid chromatography (HPLC) and the like. Also， in other embodiments， various techniques for the identification and quantification of chemical reaction products， such as proton and carbon-13 nuclear magnetic resonance (¹H and ¹³C NMR) ， infrared and ultraviolet spectroscopy (IR and UV) ， X-ray crystallography， elemental analysis (EA) ， HPLC and mass spectroscopy (MS) are used as well.

Unless otherwise indicated， in some embodiments， methods of mass spectroscopy， NMR， HPLC， protein chemistry， biochemistry， recombinant DNA techniques and pharmacology are employed.

The abbreviations used herein have the following meanings

Synthesis Examples

All the reagents were purchased commercially and used without further purification， unless otherwise stated. All yields refer to chromatographic yields. Anhydrous dimethyl formamide (DMF) was distilled from calcium hydride. Brine refers to a saturated solution of sodium chloride in distilled water. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm Yantai silica gel plates (HSGF254) using UV light as visualizing agent. Flash column chromatography was carried out using Yantai silica gel (ZCX-II， particle size 0.048-0.075 mm) . 1H-NMR and 13C-NMR spectra were recorded on a Bruker Advance 400 (1H： 400 MHz， 13C： 100 MHz ) or Bruker Advance 300 (¹H： 300 MHz， ¹³C： 75 MHz) spectrometer at ambient temperature with chemical shift values in ppm relative to TMS (δ_H 0.00 and δ_C 0.00) ， dimethyl sulfoxide (δ_H 2.50 and δ_C 39.52) ， or methanol (δ_H 3.31 and δ_C 49.00) as standard. Data are reported as follows： chemical shift， multiplicity (s＝singlet， d ＝ doublet， t ＝ triplet， q ＝ quartet， br ＝ broad， m ＝ multiplet) ， coupling constants and number of protons. HR-MS were obtained using Bruker Apex IV RTMS. Purity of compounds was determined by HPLC chromatograms acquired on an Agilent 1200 HPLC or 1260 HPLC. Analyses were conducted by an Agilent PN959990-902 Eclipse Plus C18 250 mm×4.6 mm column， using a water-MeCN gradient with MeCN from 50％to 98％or 50％to 65％in 10 min. Detection was at 254 nm， and the average peak area was used to determine purity. All the compounds were determined to be ＞95％pure.

Example 1 Synthesis of

N- (2- (3- (3-acrylamidopropanamido) phenylamino) pyrimidin-5-yl) -2-methyl-5- (3- (trifl uoromethyl) benzamido) benzamide (3) .

A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with 3- (tert-butoxycarbonylamino) propanoic acid in step 1， Example 4 to produce the title compound as white solids.

¹H NMR (400 MHz， DMSO) δ ＝ 10.58 (s， 1H) ， 10.42 (s， 1H) ， 9.90 (s， 1H) ， 9.63 (s， 1H) ， 8.80 (s， 2H) ， 8.35 -8.17 (m， 3H) ， 8.06 -7.74 (m， 5H) ， 7.32 (t， J＝9.1， 3H) ， 7.16 (t， J＝8.1， 1H) ， 6.23 (dd， J＝17.1， 10.1， 1H) ， 6.07 (dd， J＝17.1， 2.3， 1H) ， 5.57 -5.51 (m， 1H) ， 3.41 (dd， J＝12.6， 6.5， 2H) ， 2.52 (dd， J＝12.3， 5.5， 2H) ，， 2.38 (s， 3H) . ¹³C NMR (101 MHz， DMSO) δ ＝ 169.74， 167.99， 165.12， 164.40， 157.00， 150.25， 141.33， 139.82， 136.97， 135.93， 132.30， 132.22， 131.46， 131.33， 130.29， 129.82， 129.50， 128.96， 128.73， 126.77， 125.78， 125.44， 124.59， 123.07， 122.23， 119.70， 114.15， 112.91， 109.80， 36.66， 35.53， 19.28. HRMS-ESI calcd， for C₃₂H₂₈F₃N₇NaO₄ [M+Na⁺] ： 654.2053； Found： 654.2058.

Example 2 Synthesis of

(S) -l-acryloyl-N- (3- ( (5- (2-methyl-5- (3- (trifluoromethyl) benzamido) benzamido) pyrim idin-2-yl) amino) phenyl) pyrrolidine-2-carboxamide (4) .

A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -1- (tert-butoxycarbonyl) pyrrolidine-2-carboxylic acid in step 1， Example 4 to produce the title compound as white solids.

¹H NMR (400 MHz， DMSO) δ ＝ 10.59 (s， iH) ， 10.43 (s， 1H) ， 10.08 (d， J＝51.0， 1H) ， 9.65 (d， J＝10.3， 1H) ， 8.81 (s， 2H) ， 8.37 -8.23 (m， 2H) ， 8.05 -7.95 (m， 2H) ， 7.93 (s， 1H) ， 7.89-7.73 (m， 2H) ， 7.41 -7.25 (m， 3H) ， 7.18 (q， J＝7.7， 1H) ， 6.49 (ddd， J＝125.7， 16.8， 10.2， 1H) ， 6.13 (d， J＝16.6， 1H) ， 5.67 (dd， J＝21.3， 11.0， 1H) ， 4.61 (dd， J＝57.6， 5.2， 1H) ， 3.65 (dd， J＝29.9， 21.2， 2H) ， 2.38 (s， 3H) ， 2.04 (dd， J＝37.3， 30.3， 2H) ， 1.91 (s， 2H) . ¹³C NMR (101 MHz， DMSO) δ ＝ 163.20， 163.13， 160.23， 156.65， 156.30， 156.02， 149.23， 142.50， 133.65， 133.57， 132.03， 129.21， 128.17， 124.55， 123.69， 123.59， 122.53， 122.14， 122.06， 121.74， 121.67， 121.60， 121.22， 120.97， 119.90， 119.02， 118.02， 116.87， 116.84， 115.31， 115.26， 114.48， 111.96， 106.73， 106.42， 105.28， 105.10， 102.16， 102.01， 52.88， 52.62， 39.70， 39.44， 24.65， 22.18， 17.11， 14.96， 11.52. HRMS-ESI calcd， for C₃₄H₃₀F₃N₇NaO₄ [M+Na⁺] ： 680.2209； Found： 680.2204.

Example 3 Synthesis of

(S) -N- (2- ( (3- (2-acrylamidopropanamido) phenyl) amino) pyrimidin-5-yl) -2-methyl-5- (3 - (trifluoromethyl) benzamido) benzamide (5) .

A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -2- (tert-butoxycarbonylamino) propanoic acid in step 1， Example 4 to produce the title compound as white solids.

¹H NMR (400 MHz， DMSO) δ ＝ 10.57 (s， 1H) ， 10.42 (s， 1H) ， 10.04 (s， 1H) ， 9.65 (s， 1H) ， 8.81 (s， 2H) ， 8.42 (d， J＝7.4， 1H) ， 8.35 -8.23 (m， 2H) ， 8.10-7.74 (m， 5H) ， 7.26 (dq， J＝49.2， 8.1， 4H) ， 6.36 (dd， J＝17.1， 10.2， 1H) ， 6.10 (dd， J＝17.1， 2.1， 1H) ， 5.60 (dd， J＝10.2， 2.1， 1H) ， 4.56 (p， J＝7.0， 1H) ，， 2.35 (d， J＝22.6， 3H) ， 1.32 (d， J＝7.0， 3H) . ¹³C NMR (101 MHz， DMSO) δ ＝ 171.56， 167.99， 164.69， 164.41， 156.98， 150.23， 141.38， 139.63， 136.97， 135.93， 132.30， 131.95， 131.45， 131.33， 130.29， 129.81， 129.50， 129.02， 126.81， 125.95， 124.60， 123.07， 122.24， 119.71， 114.30， 112.90， 109.87， 49.42， 19.27， 18.78. HRMS-ESI calcd， for C₃₂H₂₈F₃N₇NaO₄ [M+Na⁺] ： 654.2053； Found： 654.2058.

Example 4 Synthesis of

(S) -N- (2- ( (3- (2-acrylamido-4-methylpentanamido) phenyl) amino) pyrimidin-5-yl) -2-m ethyl-5- (3- (trifluoromethyl) benzamido) benzamide (6) .

Step 1：

N- (2- ( (3-aminophenyl) amino) pyrimidin-5-yl) -2-methyl-5- (3- (trifluoromethyl) benzam ido) benzamide (Intermediate 1) (0.101g， 0.2mmol) was dissolved in 2ml dry DMF， cooled to 0℃， followed by addition of Et₃N (0.14ml， lmmol) ， HOBt (0.032g， 0.24mmol) ， N- (tert-Butoxycarbonyl) -L-leucine (0.069g， 0.3mmol) ， and slowly EDCI (0.077g， 0.4mmol) at 0℃. The reaction was allowed to room temperature slowly and reacted overnight. After stopping the reaction， the volatile components were removed under reduced pressure. The residue was diluted with saturated aqueous sodium bicarbonate and ethyl acetate. The organic phase was washed with brine， dried over anhydrous sodium sulfate， concentrated and purified by column chromatography (gradient： 30-50％ EtOAc in hexanes) to yield (S) -tert-butyl 4-methyl-1- (3 - (5 - (2-methyl-5- (3- (trifluoromethyl) benzamido) benzamido) pyrimidin-2 -ylamino) phenylamino) -1-oxopentan-2-ylcarbamate (0.105g) as light yellow solids.

Step2：

(S) -tert-butyl

4-methyl-1- (3- (5- (2-methyl-5- (3- (trifluoromethyl) benzamido) benzamido) pyrimidin-2 -ylamino) phenylamino) -1-oxopentan-2-ylcarbamate (0.105g， 0.15mmol) was dissolved in 6ml DCM， treated with 3ml TFA. The mixture was stirred at room temperature for 2 h and concentrated in vacuum. This crude product (about 0.17mmol) was dissolved in 2ml THF， cooled to 0℃， added DIEA (56uL， 0.34mmol) ， lml water， and followed by a slow addition of acryloyl chloride (21uL， 0.25mmol) . The reaction was allowed to room temperature， reacted for 2 hours. After stopping reaction， the volatile components were removed under reduced pressure. The residue was diluted with saturated aqueous sodium bicarbonate and ethyl acetate. The organic phase was washed with brine， dried over anhydrous sodium sulfate， concentrated and purified by column chromatography (gradient： 30-60％ EtOAc in hexanes) to yield (S) -N- (2- ( (3- (2-acrylamido-4-methylpentanamido) phenyl) amino) pyrimidin-5-yl) -2-m ethyl-5- (3- (trifluoromethyl) benzamido) benzamide (6) (40.8mg， 30％over three steps) as white solids.

¹H NMR (400 MHz， DMSO) δ ＝ 10.60 (d， J＝19.5， 1H) ， 10.42 (s， 1H) ， 10.13 (s， 1H) ， 9.64 (s， 1H) ， 8.81 (s， 2H) ， 8.44 -8.19 (m， 3H) ， 8.17 -7.73 (m， 5H) ， 7.43 -7.08 (m， 4H) ， 6.36 (dd， J＝17.1， 10.2， 1H) ， 6.10 (dd， J＝17.1， 2.1， 1H) ， 5.60 (dd， J＝10.2， 2.1， 1H) ， 4.61 (dd， J＝14.2， 8.6， 1H) ， 2.44 -2.28 (m， 3H) ， 1.56 (ddt， J＝35.0， 22.0， 6.9， 3H) ， 0.91 (dd， J＝ll. 7， 6.4， 6H) . ¹³C NMR (100 MHz， DMSO) δ ＝ 171.51， 167.99， 164.89， 164.41， 156.99， 150.25， 141.35， 139.62， 136.98， 135.93， 132.31， 131.95， 131.46， 131.34， 130.29， 129.82， 129.51， 129.01， 128.73， 126.80， 125.99， 125.75， 124.63， 123.07， 123.03， 122.25， 119.72， 114.34， 113.02， 109.97， 52.32， 41.51， 24.86， 23.45， 22.12， 19.27. HRMS-ESI calcd， for C₃₅H₃₄F₃N₇NaO₄ [M+Na⁺] ： 696.2522； Found： 696.2516.

Example 5 Synthesis of

(S) -N- (2- ( (3- (2-acrylamido-3-phenylpropanamido) phenyl) amino) pyrimidin-5-yl) -2-m ethyl-5- (3- (trifluoromethyl) benzamido) benzamide (7) .

A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -2- (tert-butoxycarbonylamino) -3-phenylpropanoic acid in step 1， Example 4 to produce the title compound as white solids.

¹H NMR (400 MHz， DMSO) δ ＝ 10.58 (s， 1H) ， 10.43 (s， 1H) ， 10.19 (s， 1H) ， 9.67 (s， 1H) ， 8.81 (s， 2H) ， 8.54 (d， J＝8.4， 1H) ， 8.37 -8.20 (m， 2H) ， 7.98 (dd， J＝24.2， 16.3， 3H) ， 7.88 -7.74 (m， 2H) ， 7.43 -7.09 (m， 9H) ， 6.31 (dd， J＝17.0， 10.3， 1H) ， 6.03 (d， J＝17.1， 1H) ， 5.56 (d， J＝10.3， 1H) ， 4.81 (d， J＝4.7， 1H) ， 3.07 (dd， J＝13.6， 4.4， 1H) ， 2.88 (dd， J＝13.3， 9.9， 1H) ， 2.32 (d， J＝48.8， 3H) . ¹³C NMR (101 MHz， DMSO) δ ＝170.53， 168.00， 164.91， 164.41， 156.97， 150.23， 141.39， 139.45， 138.14， 136.98， 135.93， 132.31， 131.85， 131.46， 131.34， 130.29， 129.68， 129.64， 129.47， 129.04， 128.53， 126.83， 126.08， 124.64， 119.72， 114.41， 113.04， 109.97， 55.30， 38.30， 19.28. HRMS-ESI calcd， for C₃₈H₃₂F₃N₇NaO₄ [M+Na⁺] ： 730.2366； Found： 730.2366.

Example 6 Synthesis of

(S) -N- (2- ( (3- (2-acrylamido-3-hydroxypropanamido) phenyl) amino) pyrimidin-5-yl) -2-methyl-5- (3- (trifluoromethyl) benzamido) benzamide (8) .

A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -2- (tert-butoxycarbonylamino) -3-hydroxypropanoic acid in step 1， Example 4 to produce the title compound as light yellow solids.

¹H NMR (400 MHz， DMSO) δ ＝ 10.58 (s， 1H) ， 10.43 (s， 1H) ， 10.05 (s， 1H) ， 9.65 (s， 1H) ， 8.81 (s， 2H) ， 8.30 (dd， J＝12.8， 7.6， 3H) ， 8.09 (s， 1H) ， 7.98 (d， J＝7.5， 1H) ， 7.93 (s， 1H) ， 7.89 -7.69 (m， 2H) ， 7.33 (dd， J＝15.7， 7.4， 3H) ， 7.18 (t， J＝8.1， 1H) ， 6.43 (dd， J＝17.1， 10.2， 1H) ， 6.11 (d， J＝17.3， 1H) ， 5.61 (d， J＝ll. 3， 1H) ， 5.06 (t， J＝5.2， 1H) ， 4.60 (dd， J＝13.1， 5.9， 1H) ， 3.66 (t， J＝5.3， 2H) ， 2.38 (s， 3H) . ¹³C NMR (101 MHz， DMSO) δ ＝ 169.32， 168.00， 164.98， 164.42， 156.99， 150.24， 141.34， 139.60， 136.97， 135.93， 132.31， 132.06， 131.46， 131.34， 130.29， 129.83， 129.51， 128.96， 128.73， 126.79， 125.97， 125.78， 124.63， 124.60， 123.07， 122.24， 119.72， 114.35， 112.99， 109.96， 62.26， 56.25， 19.27. HRMS-ESI calcd， for C₃₂H₂₈F₃N₇NaO₅ [M+Na⁺] ： 670.2002； Found： 670.1999.

Example 7 Synthesis of

(S) -2-acrylamido-N1- (3 - ( (5- (2-methyl-5- (3 - (trifluoromethyl) benzamido) benzamido) p yrimidin-2-yl) amino) phenyl) succinamide (9) .

A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -4-amino-2- (tert-butoxycarbonylamino) -4-oxobutanoic acid in step 1， Example 4 to produce the title compound as light yellow solids.

¹H NMR (400 MHz， DMSO) δ ＝ 10.68 (s， 1H) ， 10.47 (s， 1H) ， 10.08 (s， 1H) ， 9.62 (s， 1H) ， 8.81 (s， 2H) ， 8.49 (s， 1H) ， 8.32 (d， J＝ll. 1， 2H) ， 8.06 (s， 1H) ， 7.97 (d， J＝12.3， 2H) ， 7.86 (d， J＝8.4， 1H) ， 7.79 (t， J＝7.8， 1H) ， 7.42 (s， 1H) ， 7.33 (t， J＝9.6， 2H) ， 7.27 (d， J＝7.8， 1H) ， 7.16 (t， J＝8.0， 1H) ， 6.92 (s， 1H) ， 6.34 (dd， J＝17.1， 10.1， 1H) ， 6.10 (d， J＝17.1， 1H) ， 5.61 (d， J＝10.1， 1H) ， 4.80 (d， J＝6.1， 1H) ， 2.60 (dd， J＝15.1， 6.1， 2H) ， 2.38 (s， 3H) . ¹³C NMR (101 MHz， DMSO) δ ＝ 171.57， 170.14， 168.04， 164.92， 164.47， 157.01， 150.30， 141.32， 139.63， 136.95， 135.93， 132.31， 131.91， 131.48， 131.39， 130.32， 129.84， 129.52， 128.98， 128.75， 126.77， 126.16， 125.70， 124.64， 123.08， 122.28， 119.74， 114.39， 113.07， 110.09， 67.48， 51.19， 37.69， 25.58， 19.28. HRMS-ESI calcd， for C₃₃H₃₀F₃N₈O₅ [M+H⁺] ： 675.2291； Found： 675.2286.

Example 8 Synthesis of

(S) -4-acrylamido-5- ( (3 - ( (5- (2-methyl-5- (3- (trifluoromethyl) benzamido) benzamido) py rimidin-2-yl) amino) phenyl) amino) -5-oxopentanoic acid (10) .

A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -2- (tert-butoxycarbonylamino) pentanedioic acid in step 1， Example 4 to produce the title compound as light yellow solids.

¹H NMR (400 MHz， DMSO) δ ＝ 12.14 (s， 1H) ， 10.58 (s， 1H) ， 10.42 (s， 1H) ， 10.10 (s， 1H) ， 9.65 (s， 1H) ， 8.80 (s， 2H) ， 8.42 (d， J＝7.6， 1H) ， 8.32 (s， 1H) ， 8.28 (d， J＝7.8， 1H) ， 8.04 (s， 1H) ， 7.98 (d， J＝7.4， 1H) ， 7.92 (s， 1H) ， 7.84 (d， J＝8.3， 1H) ， 7.79 (t， J＝7.7， 1H) ， 7.35 (dd， J＝15.5， 8.1， 2H) ， 7.28 (d， J＝7.6， 1H) ， 7.18 (t， J＝8.2， 1H) ， 6.37 (dd， J＝16.9， 10.2， 1H) ， 6.11 (d， J＝17.1， 1H) ， 5.62 (d， J＝10.3， 1H) ， 4.60-4.48 (m， 1H) ， 2.38 (s， 3H) ， 2.34 -2.19 (m， 2H) ， 2.04 -1.79 (m， 2H) . ¹³C NMR (101 MHz， DMSO) δ ＝ 174.42， 170.53， 168.03， 165.09， 164.46， 156.98， 150.29， 141.39， 139.46， 136.96， 135.93， 132.31， 131.83， 131.48， 131.39， 130.32， 129.84， 129.52， 129.08， 128.78， 126.81， 126.22， 125.78， 124.64， 123.08， 122.28， 119.74， 114.44， 113.04， 109.99， 53.33， 31.04， 28.12， 19.29. HRMS-ESI calcd， for C₃₄H₃₁F₃N₇O₆ [M+H⁺] ： 690.2288； Found： 690.2283.

Example 9 Synthesis of

(S) -N- (2- ( (3- (2-acrylamido-6-aminohexanamido) phenyl) amino) pyrimidin-5-yl) -2-met hyl-5- (3- (trifluoromethyl) benzamido) benzamide trifluoroacetate (11)

Step 1：

N- (2- ( (3-aminophenyl) amino) pyrimidin-5-yl) -2-methyl-5- (3- (trifluoromethyl) benzam ido) benzamide (Intermediate 1) (0.07g， 0.14mmol) ， Nα- [ (9H-fluoren-9-ylmethoxy) carbonyl] -Nε- (tert-butoxycarbonyl) -L-lysine (0.10 lg， 0.21mmol) ， HATU (0.105g， 0.28mmol) were dissolved in dry DMF， cooled to 0℃， then added DIEA (46uL， 0.28mmol) . The reaction was allowed to room temperature slowly and reacted overnight. While stopping the reaction， the volatile components were removed under reduced pressure. The residue was diluted with saturated aqueous sodium bicarbonate and ethyl acetate. The organic phase was washed with brine， dried over anhydrous sodium sulfate， concentrated and purified by column chromatography (gradient： 30-60％ EtOAc in hexanes) to yield (S) - (9H-fluoren-9-yl) methyl tert-butyl (6- ( (3 - ( (5- (2-methyl-5- (3 - (trifluoromethyl) benzamido) benzamido) pyrimidin-2-yl) ami no) phenyl) amino) -6-oxohexane-1， 5-diyl) dicarbamate (0.128g， 96％) as yellow solids.

Step2：

(S) - (9H-fluoren-9-yl) methyl tert-butyl (6- ( (3 - ( (5- (2-methyl-5- (3- (trifluoromethyl) benzamido) benzamido) pyrimidin-2-yl) ami no)phenyl) amino) -6-oxohexane-1， 5-diyl) dicarbamate (0.128g， 0.13mmol) was dissolved in lml dry DMF， treated with lml morpholine， reacted at room temperature for 3 hours. While stopping the reaction， the volatile components were removed under reduced pressure. The residue was diluted with saturated aqueous sodium bicarbonate and ethyl acetate. The organic phase was washed with brine， dried over anhydrous sodium sulfate， concentrated and purified by column chromatography (gradient： 2-4％MeOH in DCM) to yield (S) -tert-butyl 5-amino-6- (3- (5- (2-methyl-5- (3- (trifluoromethyl) benzamido) benzamido) pyrimidin-2-ylamino) phenylamino) -6-oxohexylcarbamate (0.084g， 88％) as light yellow solids.

Step3：

(S) -tert-butyl

5-amino-6- (3- (5- (2-methyl-5- (3- (trifluoromethyl) benzamido) benzamido) pyrimidin-2-ylamino) phenylamino) -6-oxohexylcarbamate (0.084g， 0.114mmol) was dissolved in 1ml THF， cooled to 0℃， added DIEA (38uL， 0.23mmol) ， 0.5ml water， and followed by a slow addition of acryloyl chloride (18uL， 0.23mmol) . The reaction was allowed to room temperature， reacted for 2 hours. While stopping reaction， the volatile components were removed under reduced pressure. The residue was diluted with saturated aqueous sodium bicarbonate and ethyl acetate. The organic phase was washed with brine， dried over anhydrous sodium sulfate， concentrated， yielding crude product (0.080g) as yellow solids. The latter was dissolved in 1ml DCM， treated with 0.5ml TFA. The mixture was stirred at room temperature for 2 h and concentrated in vacuum， the obtained solids was washed with diethyl ether， then gained the title compound (11) (0.037 g， 47％over 2 steps) as yellow solids.

¹H NMR (400 MHz， DMSO) δ ＝ 10.60 (s， 1H) ， 10.44 (s， 1H) ， 10.11 (s， 1H) ， 9.64 (s， 1H) ， 8.80 (s， 2H) ， 8.39 (d， J＝7.9， 1H) ， 8.31 (s， 1H) ， 8.27 (d， J＝7.9， 2H) ， 8.04 (s， 1H) ， 7.97 (d， J＝7.8， 2H) ， 7.93 (d， J＝2.1， 1H) ， 7.88 -7.75 (m， 3H) ， 7.37 -7.26 (m， 4H) ， 7.17 (t， J＝8.1， 1H) ， 6.37 (dd， J＝17.1， 10.2， 1H) ， 6.10 (dd， J＝17.1， 2.1， 1H) ， 5.60 (dd， J＝10.2， 2.1， 1H) ， 4.51 (dd， J＝13.8， 8.3， 1H) ， 2.35 (d， J＝22.7， 3H) ， 1.76-1.53 (m， 2H) ， 1.35 (dd， J＝16.0， 10.2， 4H) . ¹³C NMR (75 MHz， DMSO) δ ＝ 171.19， 168.02， 164.99， 164.45， 156.97， 150.26， 141.37， 139.60， 136.96， 135.92， 132.32， 131.94， 131.49， 131.38， 130.32， 129.88， 129.45， 129.06， 128.75， 126.80， 126.18， 126.04， 124.65， 122.62， 122.27， 119.74， 114.32， 112.94， 109.89， 100.00， 53.92， 41.71， 32.97， 32.58， 23.36， 19.29. HRMS-ESI calcd， for C₃₅H₃₆F₃N₈O₄ [M+H⁺] ： 689.2812； Found： 689.2807.

Example 10 Synthesis of

(S) -N- (2- ( (3- (2-acrylamido-3- (1H-imidazol-5-yl) propanamido) phenyl) amino) pyrimid in-5-yl) -2-methyl-5- (3- (trifluoromethyl) benzamido) benzamide (12) .

A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -2- (tert-butoxycarbonylamino) -3- (1H-imidazol-5-yl) propanoic acid in step 1， Example 4 to produce the title compound as light yellow solids.

^tH NMR (400 MHz， DMSO) δ ＝ 10.70 (s， 1H) ， 10.48 (s， 1H) ， 10.23 (s， 1H) ， 9.63 (s， 1H) ， 8.81 (s， 2H) ， 8.59 (d， J＝7.6， 1H) ， 8.32 (d， J＝8.7， 2H) ， 8.06 (s， 1H) ， 7.95 (t， J＝11.9， 3H) ， 7.87 (d， J＝8.2， 1H) ， 7.79 (t， J＝7.5， 1H) ， 7.42 -7.22 (m， 3H) ， 7.17 (t， J＝8.1， 1H) ， 6.96 (s， 1H) ， 6.36 (dd， J＝17.4， 10.1， 1H) ， 6.08 (d， J＝16.9， 1H) ， 5.60 (d， J＝10.2， 1H) ， 4.79 (d， J＝6.1， 1H) ， 3.11 -2.83 (m， 2H) ， 2.38 (s， 3H) . ¹³C NMR (101 MHz， DMSO) δ ＝ 170.08， 167.97， 164.91， 164.37， 156.90， 150.23， 141.27， 139.42， 136.92， 135.83， 134.67， 132.29， 131.82， 131.33， 131.26， 130.20， 129.66， 129.40， 128.90， 128.67， 126.74， 126.03， 125.68， 124.65， 122.24， 119.74， 114.38， 113.02， 110.05， 53.87， 29.41， 19.23. HRMS-ESI calcd， for C₃₅H₃₁F₃N₉O₄ [M+H⁺] ： 698.2451； Found： 698.2447.

Example 11

(S) -N- (2- ( (3- (2-acrylamido-6- (pent-4-ynamido) hexanamido) phenyl) amino) pyrimidin-5-yl) -2-methyl-5- (3- (trifluoromethyl) benzamido) benzamide (13) .

A procedure similar to Example 12 was repeated except for replacing

FL with pent-4-ynoic acid to produce the title compound as light yellow solids.

¹H NMR (400 MHz， DMSO) δ ＝ 10.58 (s， 1H) ， 10.42 (s， 1H) ， 10.09 (s， 1H) ， 9.64 (s， 1H) ， 8.81 (s， 2H) ， 8.41 -8.23 (m， 3H) ， 8.08 -7.90 (m， 3H) ， 7.90 -7.74 (m， 3H) ， 7.44 - 7.25 (m， 3H) ， 7.18 (t， J＝8.1， 1H) ， 6.39 (dd， J＝17.1， 10.2， 1H) ， 6.11 (dd， J＝17.1， 2.0， 1H) ， 5.61 (dd， J＝10.2， 2.0， 1H) ， 4.52 (dd， J＝13.5， 8.4， 1H) ， 3.09 -2.96 (m， 2H) ， 2.72 (t， J＝2.5， 1H) ， 2.39 (s， 3H) ， 2.37 -2.28 (m， 2H) ， 2.23 (t， J＝7.0， 2H) ， 1.79-1.57 (m， 2H) ， 1.50 -1.16 (m， 4H) . ¹³C NMR (101 MHz， DMSO) 5 ＝ 171.11， 170.52， 168.02， 165.00， 164.43， 157.00， 150.27， 141.39， 139.59， 136.98， 135.95， 132.31， 131.97， 131.47， 131.36， 130.30， 129.85， 129.53， 129.04， 128.77， 126.81， 126.00， 125.79， 124.61， 123.06， 122.26， 119.74， 114.35， 112.98， 109.94， 84.23， 71.69， 53.80， 38.81， 34.69， 32.41， 29.31， 23.40， 19.28， 14.75. HRMS-ESI calcd， for C₄₀H₃₉F₃N_sNaO₅ [M+Na⁺] ： 791.2893； Found： 791.2889.

Example 12

(S) -3- (3 - (5 -acrylamido-6- (3 - (5- (2-methyl-5- (3 - (trifluoromethyl) benzamido) benzamid o) pyrimidin-2-ylamino) phenylamino) -6-oxohexylamino) -3-oxopropyl) -5， 5-difluoro-7 ， 9-dimethyl-5H-dipyrrolo [1， 2-c： 1′ ， 2′-f] [1， 3， 2] diazaborinin-4-ium-5-uide (14)

Compound (11) (0.038g， 0.055mmol) ， 4， 4-difluoro-5， 7-dimethyl-4-bora-3a， 4a-diaza- (S) -indacene-3-propionic acid (

FL) (0.015g， 0.05mmol) ， HATU (0.038g， 0.1mmol) were dissolved in dry DMF， cooled to 0℃， then added DIEA (41uL， 0.25mmol) . The reaction was allowed to room temperature slowly and reacted overnight. While stopping the reaction， the volatile components were removed under reduced pressure. The residue was diluted with saturated aqueous sodium bicarbonate and ethyl acetate. The organic phase was washed with brine， dried over anhydrous sodium sulfate， concentrated and purified by column chromatography (gradient： 60％EtOAc in hexanes-1％MeOH in EtOAc) to yield the designed product (14) (0.033g， 70％) as red solids.

¹H NMR (400 MHz， MeOD) δ ＝ 8.77 (s， 2H) ， 8.28 (s， 1H) ， 8.22 (d， J＝7.9， 1H) ， 8.08 (s， 1H) ， 7.95 (d， J＝2.0， 1H) ， 7.90 (d， J＝7.7， 1H) ， 7.75 (d， J＝7.8， 1H) ， 7.73 -7.66 (m， 1H) ， 7.38 (s， 1H) ， 7.34 (t， J＝6.6， 2H) ， 7.24 (dt， J＝15.7， 8.0， 2H) ， 6.97 (d， J＝3.9， 1H) ， 6.38 (dd， J＝17.1， 10.1， 1H) ， 6.25 (dd， J＝17.1， 1.7， 2H) ， 6.18 (s， 1H) ， 5.69 (dd， J＝10.2， 1.6， 1H) ， 4.58 -4.49 (m， 1H) ， 3.35 (s， 2H) ， 3.20 (dd， J＝13.5， 6.6， 4H) ， 2.57 (t， J＝7.6， 2H) ， 2.49 (s， 3H) ， 2.47 (s， 3H) ， 2.26 (s， 3H) ， 1.95 -1.85 (m， 1H) ， 1.80 (m， 1H) ， 1.55 (dd， J＝13.6， 6.6， 2H) ， 1.50-1.37 (m， 2H) ， 1.36 -1.27 (m， 2H) . ¹³C NMR (101 MHz， MeOD) δ ＝ 165.68， 163.88， 163.43， 162.32， 161.59， 158.86， 157.86， 155.22， 152.07， 149.34， 142.68， 136.58， 132.94， 130.70， 128.40， 128.34， 127.89， 127.18， 125.67， 124.41， 123.31， 123.18， 123.01， 122.48， 121.53， 120.76， 120.46， 120.29， 118.15， 117.46， 116.56， 116.36， 114.87， 112.13， 111.91， 108.47， 107.12， 105.99， 102.99， 46.35， 30.86， 26.82， 23.80， 20.86， 16.47， 15.01， 10.09， 5.67， 2.00. HRMS-ESI calcd. for C₄₉H₄₈BF₅N₁₀NaO₅ [M+Na⁺] ： 985.3720； Found： 985.3717.

Biological assays

Kinase enzymology assays

Kinases were purchased from Carna Biosciences. Kinase enzymology assays were performed according to the protocols specified for the

KinEase^TM assays sold by Cisbio Bioassays.

Kinetic study of Btk inhibitors

The kinase assays were performed at room temperature. The compounds with serial dilution in DMSO were added into reaction buffer with 0.5nM Btk， incubating with different periods of time (0min， 4min， 8min， 12min， 16min， and 20min) . Enzyme reaction was started by adding ATP and substrate to the reaction mixture. The enzyme activity was measured with

KinEase^TM assays. The data analysis was guided by the book， Enzyme Kinetics by Hans Bisswanger [Bisswanger， H. Enzyme Kinetics -principles and methods， 103-106 (Weinheim， 2002) ] .

Recombinant protein labeling assays

In 25 μL of PBS buffer， 0.5 ug of recombinant Btk was incubated with increasing concentrations of probe 14 for 2 h and then analyzed by SDS/PAGE and fluorescent gel scanning (fluorescence， CY2) . The gel was then blotted， and the total Btk levels were detected by standard silver staining. The concentration course labeling procedure was similar to that for the time course labeling.

Cellular labeling assays

Labeling of Btk by probe 14. A total of 1.5× 10⁶ cells were treated with probe 14 at 1 μM for different lengths of times (5 min， 10 min， 20 min， 30 min， 1 h， 2 h， 3 h， or 4 h) ， washed， lysed in cell lysis buffer (Beyotime) containing 1 mM PMSF and 10 mM NaF (Invitrogen) ， and centrifuged. The sample protein concentrations were quantified using a NanoDrop 2000 spectrophotometer， and the samples were adjusted to the same concentration then analyzed by SDS/PAGE and fluorescent gel scanning (fluorescence， CY2) . The gel was then blotted， and the total Btk levels were detected by a standard Western blot. The concentration course labeling procedure was similar to that for the time course labeling.

Immunoprecipitation of Btk. LY7 cells were treated with probe 14 at 0.5μM for 2h， lysed in binding buffer (20 mM Na₃PO₄， pH 7.5， 150 mM NaCl) containing phosphatase inhibitors and protease inhibitors. Obtained lysates was preincubated with Protein A Sepharose beads (GE healthcare， 17-5138-01) to remove intrinsic cellular IgG proteins. Meanwhile， anti-Btk (CST， 8574S) from rabbit was preincubated with Protein A Sepharose beads for 2 hours at 4℃. Then， the pre-treated lysates were added to the pre-treated immobilized Protein A Sepharose， incubated for 2 hours at 4℃. The immune complexes were washed with binding buffer for four times and eluted with LDS sample buffer (50 mM Tris-HCl， 2％SDS， 0.1％bromophenol blue， 10％glycerol， 1％DTT) and analyzed by Western blot， as described above.

Competition assay. LY7 cells were preincubated with the compounds (1 μM) for 1 h before labeling with probe 14 under the proper time and concentration conditions. Then， the cells were lysed and analyzed as described above.

Target engagement of Btk inhibitors. LY7 cells were preincubated with different concentrations of compounds for 1 h before labeling with probe 14. Then， the samples were lysed and analyzed as described above. Gelpro32 software was used to analyze the Btk band density to obtain the half-maximum active site occupancy values.

Results Discussion

Structure-activity relationship of Btk inhibitors

The Btk inhibitor activities of the present compounds (based on the kinase enzymology assays) are shown in the following table：

*The values are determined by a single run of duplicates and all others are means of two individual measurements.

Compound 14 is a selective affinity probe for Btk

When recombinant Btk (0.5 ug) was incubated with increasing concentrations of probe 14， fluorescent signals increased accordingly， and 0.5 uM was selected as the probe concentration for the next steps because it already provided a sufficiently strong signal. When probe 14 was incubated with Btk for increasing time periods， the brightness of the fluorescent signals reached a maximum at 2 hours (Figs. 6 and 7) .

In live OCI-Ly7 cell， a dominant band was present at the expected molecular weight of Btk (approximately 76 kDa) ， with a couple of minor bands at lower molecular weights. This dominant band was also immunoreactive against anti-Btk antibody. Concentration course experiments indicated again that the Btk band intensity reached saturation at 0.5 uM probe 14. Time course experiments also suggested that an incubation time of 2 hours was sufficient for labeling (Figs 8 and 9) . To further confirm that Btk was indeed labeled by probe 14 in these cells， Btk was successfully immunoprecipitated from probe 14-labeled lysates (Fig. 10) . Taken together， Btk was indeed the dominant band labeled by probe 14 in OCI-Ly7 cells (Fig. 13a) . As expected， no significant labeling was detected in Jurkat cells， a T-cell line that does not express Btk (Fig. 13b) .

2， 5-Diaminopyrimidine series of inhibitors directly engage Btk in live cells

After the labeling conditions were optimized， it was examined whether probe 14 could be used to access the target engagement of inhibitors towards Btk. Two types of structurally different Btk inhibitors were examined： the clinically approved Btk drug ibrutinib and compound 2， which contains the same scaffold as probe 14. Cells were first incubated with the inhibitors for 1 hour， followed by 2 hours incubation with 0.5 uM of probe 14. As shown in Fig. 11， both compounds at 1 uM effectively blocked the labeling of Btk by probe 14. To measure the extent of Btk occupancy by the inhibitors in live cells， OCI-Ly7 cells were incubated with increasing concentrations of the compounds for 1 hour prior to labeling with probe 14 for 2 hours. After cell lysis， the protein contents were directly loaded onto gels. After electrophoresis， the bands’ fluorescent densities were measured. As presented in Fig. 12， the IC₅₀ values for Btk occupancy by ibrutinib and compound 2 were 2 nM and 8 nM， respectively.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes are to be included within the spirit and purview of this application and scope of the appended claims. All publications， patents， and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

A compound having a general formula (Id) ， or a pharmaceutical acceptable salt thereof：

wherein

p is 0， 1， 2 or 3；

q is 0， 1， 2 or 3； and

R₁₁ is a substituent containing an end group selected from OH， COOH， CONH₂， NH₂， and a nitrogen containing heterocycle.
The compound according to claim 1 or a pharmaceutical acceptable salt thereof， wherein p is 0 and q is 0.
The compound according to any of the preceding claims or a pharmaceutical acceptable salt thereof， wherein R₁₁ is an alkyl group substituted by a substituent selected from the group consisting of OH， COOH， CONH₂， NH₂， and a nitrogen containing heterocycle as end group， wherein one or more CH₂ moieties in said alkyl group are optionally replaced with a divalent group selected from the group consisting of-NH-， -CO-， -SO₂-and -SO-.
The compound according to any of the preceding claims or a pharmaceutical acceptable salt thereof， wherein R₁₁ is selected from the group consisting of
A compound or a pharmaceutical acceptable salt thereof， wherein said compound is selected from the group consisting of：
A pharmaceutical composition containing a therapeutically effective amount of a compound according to any of claims 1-5 or a pharmaceutical acceptable salt thereof， in combination with one or more pharmaceutically acceptable carriers.
Use of a compound according to any of claims 1-5 or a pharmaceutical acceptable salt thereof in manufacture of a medicament for treating or preventing a disease selected from the group consisting of autoimmune disease， heteroimmune disease， inflammatory disease， cancer， and thromboembolic disease.
Use of a compound according to any of claims 1-5 or a pharmaceutical acceptable salt thereof in the preparation of a Btk affinity probe.
Use of any of the following compounds or a pharmaceutical acceptable salt thereof in the preparation of a Btk affinity probe：
A Btk affinity probe as represented by the following formula (Ic) ：

wherein the Btk inhibitor moiety is derivable from a compound having a general formula (Id) ：

wherein

p is 0， 1， 2 or 3；

q is 0， 1， 2 or 3；

R₁₁ is a substituent containing an end group selected from OH， COOH， CONH₂， NH₂， and a nitrogen containing heterocycle；

wherein X and Y are independently selected from the group consisting of a bond， -O (CO) -， -NR^a (CO) -， -NR^a-，
-O-， -S-， -S-S-， -O-NR^a-， -O (CO) O-， -O (CO) NR^a-， -NR^a (CO) NR^a-， -N＝CR^a-， -S (CO) -， -S (O) -， and -S (O) ₂-；

wherein
forms a N-containing heterocycle；

R^a is hydrogen or alkyl；

wherein the linker moiety is selected from a bond， an optionally substituted alkyl moiety， an optionally substituted heterocycle moiety， an optionally substituted amide moiety， a ketone moiety， an optionally substituted carbamate moiety， an ester moiety， or a combination thereof；

wherein the reporter moiety is selected from the group consisting of a label， a dye， a photocrosslinker， a cytotoxic compound， a drug， an affinity label， a photoaffinity label， a reactive compound， an antibody or antibody fragment， a biomaterial， a nanoparticle， a spin label， a fluorophore， a metal-containing moiety， a radioactive moiety， a novel functional group， a group that covalently or noncovalently interacts with other molecules， a photocaged moiety， an actinic radiation excitable moiety， a ligand， a photoisomerizable moiety， biotin， a biotin analogue， a moiety incorporating a heavy atom， a chemically cleavable group， a photocleavable group， a redox-active agent， an isotopically labeled moiety， a biophysical probe， a phosphorescent group， a chemiluminescent group， an electron dense group， a magnetic group， an intercalating group， a chromophore， an energy transfer agent， a biologically active agent， a detectable label， or a combination thereof.
The Btk affinity probe according to claim 10， wherein p is 0 and q is 0.
The Btk affinity probe according to any of claims 10-11， wherein R₁₁ is an alkyl group substituted by a substituent selected from the group consisting of OH， COOH， CONH₂， NH₂， and a nitrogen containing heterocycle as end group， wherein one or more CH₂ moieties in said alkyl group are optionally replaced with a divalent group selected from the group consisting of-NH-， -CO-， -SO₂-and -SO-.
The Btk affinity probe according to any of claims 10-12， wherein R₁₁ is selected from the group consisting of
The Btk affinity probe according to any of claims 10-13， wherein the linker moiety is a bond.
The Btk affinity probe according to any of claims 10-14， wherein the reporter moiety is a fluorophore.
The Btk affinity probe according to claim 15， wherein the fluorophore is a Bodipy fluorophore.
The Btk affinity probe according to claim 16， wherein the Bodipy fluorophore is a Bodipy FL fluorophore.
A Btk affinity probe having a structure of
A method for assessing the efficacy of a potential Btk inhibitor in a mammal， comprising administering a potential Btk inhibitor to the mammal， administering the Btk affinity probe of any of claims 10-18 to the mammal or to cells isolated from the mammal； measuring the activity of the reporter moiety of the Btk affinity probe， and comparing the activity of the reporter moiety to a standard.
A method for assessing the pharmacodynamics of a Btk inhibitor in a mammal， comprising administering a Btk inhibitor to a plurality of mammals， administering the Btk affinity probe of any of claims 10-18 to the plurality of mammals or to cells isolated from a plurality of mammals， and measuring the activity of the Btk affinity probe at different time points following the administration of the inhibitor.
A method for in vitro labeling of a Btk enzyme comprising contacting cells or tissues expressing the Btk enzyme with the Btk affinity probe of any of claims 10-18.
A method for detecting a labeled Btk enzyme comprising separating proteins， the proteins comprising a Btk enzyme labeled by the Btk affinity probe of any of claims 10-18， by electrophoresis and detecting the Btk affinity probe by fluorescence.