CN108779078B

CN108779078B - Inhibitors and probes of kinases and uses thereof

Info

Publication number: CN108779078B
Application number: CN201580085270.XA
Authority: CN
Inventors: 潘峥婴; 左莹莹; 李锡涛
Original assignee: Beijing Reciprocapharmaceuticals Co ltd; Peking University Shenzhen Graduate School
Current assignee: Beijing Reciprocapharmaceuticals Co ltd; Peking University Shenzhen Graduate School
Priority date: 2015-10-12
Filing date: 2015-10-12
Publication date: 2021-12-31
Anticipated expiration: 2035-10-12
Also published as: CN108779078A; WO2017063103A1

Abstract

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

Description

Inhibitors and probes of kinases and uses thereof

Technical Field

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

Background

Bruton's tyrosine kinase (Btk) is a cytoplasmic non-tyrosine kinase that is expressed only in hematopoietic cells, except in natural killer and T cells. Btk is involved in several signaling pathways, particularly the B Cell Receptor (BCR) pathway, which is critical in the development and differentiation of B cells [ Mohamed, A. J. et al, Bruton's tyrosine kinase (Btk): function, regulation, and transformation with specific emophilis on the PH domain, Immunol. Rev. 228, 58-73 (2009) ].

In cells, Btk is activated by phosphorylation of its upstream kinase via a tyrosine residue (Tyr551), followed by autophosphorylation of another tyrosine residue (Tyr 223). Fully activated Btk then phosphorylates its substrates, including PLC- γ 2 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 antigens in pre-BCR and BCR signalling with freedom to cell of cells malignans, nat. rev. immunol. 13, 578-591 (2013) ].

Several Btk inhibitors have been developed (fig. 1).

Covalent irreversible inhibitor ibrutinib (CRA-032765, PCI-32765, Imbruvica) from Celera/Pharmacyclics/Janssen became the first clinically approved Btk targeted drug in 11 months of 2013 [ Pan, Z, et al, Discovery of selective ir reversible inhibitors for Bruton's tyrosine kinase. ChemMedChem. 2, 58-61 (2007) ].

CC-292 (AVL-292) from Celgene is the second covalent irreversible inhibitor currently in clinical trials [ Singh, J. et al inventor; Celgene Avilomics Research, Inc., assignee, 2, 4-diaminopyrimides usefrul as kinase inhibitors, U.S. Pat. No. 5, 8,609,679.2013,17 ].

Both ibrutinib and CC-292 form covalent bonds with cysteine residue (Cys481) of Btk at the edge of the ATP-binding pocket.

Other clinical stages of Btk inhibitors include compounds from ONO Pharmaceutical and PRN1008/HM71224 from Hanmi Pharmaceutical [ Yamamoto, S. & Yoshizawa T. inventors; Ono Pharmaceutical Co., Ltd., assignee, Purinone derivative U.S. Pat. No. US 8,940,725.2015 Jan 27; Hanmi Pharmaceutical Company Limited, Safety, PK/PD, Food Effect Study of Oralyy Administered HM71224 in health Adult Malutenters. https// clinical trials. gov/ct2/show/NCT01765 (Accessed: 478.7.4.4.d.).

Non-covalent reversible Btk inhibitor GDC-0834 from Gilead/Roche was evaluated in a phase I clinical trial, but no recent progress has been reported [ Liu, L et al, Significant sites differences in amino hydrolytics of GDC-0834, a novel and selective Bruton's tyrosine kinase inhibitor. Drug Metab. Dispos. 39, 1840-1849 (2011) ].

Target engagement (target engagement) means that the biological target is expected to be occupied by a Drug molecule [ Copeland, r. a., pomliano, d.l. & Meek, t.d. Drug-target resistance time and matters interactions for lead optimization. nat. Drug discovery discov. 5, 730-. This information is crucial to establishing correlations at the molecular level between phenotypic observations and inhibitor-biomolecule interactions. Due to their inherent reactive groups, targeted Covalent drugs are particularly suitable for The development of small molecule affinity probes [ Potashman, M.H. & Duggan, M.E. Covalent modulators: an orthogonal adaptive approach to Drug design. J. Med. chem. 52, 1231-.

PCI-33380 was designed based on The backbone of ibrutinib and has been used in cellular and in vivo studies that demonstrated a correlation between inhibitor binding events and phenotypic readouts of cellular responses due to functional inhibition of Btk [ Honigberg, L. A. et al, The Bruton type kinase inhibitor PCI-32765 blocks B-cell activation and is infection disorders in models of autoimmmune disease and B-cell magnancy, Proc.

Furthermore, the use of fluorescent probes in clinical trials plays an important role in determining the appropriate drug dose for a patient [ O' Brien, S. et al, Ibrutinib as initial therapy for electrodely Patients with chronic lymphocytic lymphoma: an open-label, multicentre, phase 1b/2 tertiary, Lancet Oncol. 15, 48-58 (2014) ].

In addition to PCI-33380, other Btk fluorescent probes that also utilize an ibrutinib scaffold have recently been reported for Btk imaging in living cells (FIG. 2) [ Turetsky, A., Kim, E., Kohler, R. H., Miller, M.A. & Weissleder, R. Single cell imaging of Bruton's type kinase using an irreversible addition. Sci. Rep. 4, 4782 (2014); Zhang, Q., Liu, H. & Pan, Z. A general improvement for the reduction of fluorescent probes above for imaging of kinases in vivo cells, Chem. 50, 15319 (2014) 15322.

As depicted in fig. 3, affinity probes typically comprise three components: a recognition group, a reactive group, and a reporter group. The recognition group introduces 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 reporter group provides a convenient means of identifying the probe-bound proteins within a complex proteome.

Figure 4 shows a general scheme of an assay to examine target binding of drug molecules. By sequentially adding the inhibitor and probe to the biological sample (cells, tissue, etc.), the intensity of the probe-labeled bands will give a direct readout of those biological targets not occupied by the inhibitor. As the concentration of inhibitor increases, a decrease in the intensity of the band indicates that a portion of the biological target is bound by the inhibitor.

Disclosure of Invention

The object of the present invention is to provide a series of novel Btk covalent inhibitors based on a 2, 5-diaminopyrimidine backbone. Another object of the invention is to develop the series of inhibitors as novel affinity Btk probes. The resulting probes are capable of selectively labeling Btk and provide an efficient method of directly measuring target engagement (target engagement) of Btk inhibitors in living cells.

One aspect is a compound having the general formula (Ia) or (Ib-1) or (Id):

wherein R is selected from the group consisting of a bond, carbonylalkyleneamino (e.g., carbonyl C)_1-6Alkyleneamino, (((azacycloalkane-2-yl) -alkyl) oxomethane) -1, N-diyl (e.g. ((C)_5-7Azacycloalkane-2-yl) -C_0-2Alkyl) oxomethane) -1, N-diyl); preferably R is selected from

、

And;

wherein R is₁Selected from the group consisting of alkyl, arylalkyl, hydroxyalkyl, aminocarbonylalkyl, carboxyalkyl, aminoalkyl and heteroarylalkyl; preferably R₁Is selected from

、

、

、

,

、

、

And

(ii) a More preferably R₁Is selected from

、

、

、

、

And

；

wherein

p is 0, 1,2 or 3, preferably 0;

q is 0, 1,2 or 3, preferably 0;

R₁₁is selected from OH, COOH, CONH₂、NH₂And a substituent of a terminal group of the nitrogen-containing heterocycle; preferably R₁₁Is selected from OH, COOH, CONH₂、NH₂And an alkyl group substituted with a nitrogen-containing heterocycle as a substituent of a terminal group, wherein one or more CH in the alkyl group₂Part being optionally selected from-NH-, -CO-, -SO₂-and-SO-divalent radical substitution; more preferably R₁₁Is selected from

、

、

、

And

。

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

One aspect is a compound of the above general formula (Ia) or (Ib-1) or (Id), or a pharmaceutically acceptable salt thereof, as Btk inhibitors for the treatment of B-cell malignancies and autoimmune diseases such as rheumatoid arthritis and lupus.

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

One aspect is a Btk affinity probe comprising a Btk inhibitor moiety, a reporter moiety, and a linker moiety linking the Btk inhibitor moiety and the reporter moiety; wherein the Btk inhibitor moiety can be derived from a compound having the general formula (Ia) or (Ib-1) or (Id) above, or a pharmaceutically acceptable salt thereof.

One aspect is the use of a compound having the general formula (Ia) or (Ib-1) or (Id) above, or a pharmaceutically acceptable salt thereof, as part of a Btk affinity probe comprising a Btk inhibitor moiety, a reporter moiety, and a linker moiety linking the Btk inhibitor moiety and the reporter moiety; wherein the Btk inhibitor moiety can be derived from a compound having the general formula (Ia) or (Ib-1) or (Id) above, or a pharmaceutically acceptable salt thereof.

One aspect is a Btk affinity probe, represented by formula (Ic) below:

wherein the Btk inhibitor moiety can be derived from a compound having the general formula (Ia) or (Ib-1) or (Id) above, 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

Forming an N-containing heterocycle;

R^ais hydrogen or alkyl.

One embodiment is a Btk affinity probe, wherein the linker moiety covalently links the Btk inhibitor moiety and the reporter moiety. Another embodiment is a Btk affinity probe, wherein the Btk inhibitor moiety modifies a cysteine residue of the Btk enzyme. Another embodiment is a Btk affinity probe, wherein the Btk inhibitor moiety covalently modifies a cysteine residue of the Btk enzyme. Yet another embodiment is a Btk affinity probe, wherein the cysteine residue is within an ATP-binding pocket of the Btk enzyme. Yet another embodiment is a Btk affinity probe, wherein the cysteine residue is Cys481 of the Btk enzyme. One embodiment is a Btk affinity probe, wherein the linker moiety is selected from a bond, an optionally substituted alkyl moiety, an optionally substituted heterocyclic moiety, an optionally substituted amide moiety, a ketone moiety, an optionally substituted carbamate moiety, an ester moiety, or a combination thereof. Another embodiment is a Btk affinity probe, wherein the linker moiety is a bond.

Also described herein are Btk affinity probes, 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 interacts covalently or non-covalently with other molecules, a photocaged moiety, an actinic radiation-labile moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analog, a moiety that incorporates a heavy atom, a chemically cleavable group, a photolyzable group, a redox 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 bioactive agent, A detectable label, or a combination thereof. Another embodiment is a Btk affinity probe, wherein the reporter moiety is a fluorophore. Yet another embodiment is a Btk affinity probe, wherein the fluorophore is a Bodipy fluorophore. Yet another embodiment is a Btk affinity probe, wherein the Bodipy fluorophore is a Bodipy FL fluorophore.

Presented herein are Btk affinity probes, wherein the Btk inhibitor moiety is derived from an irreversible inhibitor of Btk. One embodiment is a Btk affinity probe, wherein the irreversible inhibitor of Btk is;

。

another embodiment is a Btk affinity probe having the structure:

。

another embodiment is a Btk affinity probe, wherein the probe selectively labels a phosphorylated conformation of Btk. Another embodiment is a Btk affinity probe, wherein the phosphorylated conformation of Btk is an activated or non-activated form of Btk. Another embodiment is a Btk affinity probe, wherein the phosphorylated conformation of Btk is an activated form of Btk. One embodiment is a Btk affinity probe, wherein the probe is cell permeable.

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

Another aspect is a method for evaluating the pharmacodynamics of a Btk inhibitor in a mammal comprising administering the Btk inhibitor to the mammal, administering a Btk affinity probe presented herein to the mammal or to a cell isolated from the mammal; and measuring the activity of the reporter moiety of the Btk affinity probe at different time points after administration of the inhibitor.

Another aspect is a method for in vitro labeling of a Btk enzyme, comprising contacting an activated Btk enzyme with a Btk affinity probe described herein. One embodiment is a method for in vitro labeling of a Btk enzyme, wherein the contacting step comprises incubating the activated Btk enzyme with a Btk affinity probe as presented herein.

Another aspect is a method of in vitro labeling of a Btk enzyme comprising contacting a cell or tissue expressing the Btk enzyme with a Btk affinity probe described herein.

One aspect is a method for detecting a labeled Btk enzyme comprising separating a protein comprising a Btk enzyme labeled with a Btk affinity probe described herein by electrophoresis and detecting the Btk affinity probe by fluorescence.

The invention comprises the following technical scheme:

technical solution 1

A compound having the general formula (Ia) or (Ib-1) or (Id):

wherein R is selected from the group consisting of a bond, carbonylalkyleneamino, ((((azacycloalkane-2-yl) -alkyl) oxomethane) -1, N-diyl; preferably R is selected from (((C)_5-7Azacycloalkane-2-yl) -C_0-2Alkyl) oxomethane) -1, N-diyl);

wherein R is₁Selected from the group consisting of alkyl, arylalkyl, hydroxyalkyl, aminocarbonylalkyl, carboxyalkyl, aminoalkyl and heteroarylalkyl; preferably R₁Selected from the group consisting of arylalkyl, hydroxyalkyl, aminocarbonylalkyl, carboxyalkyl, aminoalkyl and heteroarylalkyl; or preferably R₁Is selected from

、

、

、

、

And

(ii) a Or preferably R₁Is selected from

、

、

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 pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

。

technical solution 3

A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1-2, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

Technical solution 4

Use of a compound according to any one of claims 1-2 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a disease selected from the group consisting of autoimmune diseases, heteroimmune diseases, inflammatory diseases, cancer, and thromboembolic diseases.

Technical solution 5

Use of a compound according to

claim

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

Technical scheme 6

The use of any one of the following compounds in the preparation of a Btk affinity probe:

。

technical scheme 7

A Btk affinity probe of formula (Ic) below:

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

wherein R is selected from the group consisting of a bond, carbonylalkyleneamino, ((((azacycloalkane-2-yl) -alkyl) oxomethane) -1, N-dimethyl; preferably R is selected from (((C)_5-7Azacycloalkane-2-yl) -C_0-2Alkyl) oxomethane) -1, N-dimethyl);

、

、

、

、

And

(ii) a Or preferably R₁Is selected from

、

、

And

；

wherein

p is 0, 1,2 or 3, preferably 0;

q is 0, 1,2 or 3, preferably 0;

R₁₁is selected from OH, COOH, CONH₂、NH₂And substituents of terminal groups of nitrogen-containing heterocycles (e.g. as defined by the formula R)₁₁Derivatization of the terminal group(s) by removal of hydrogen atoms); preferably R₁₁Is selected from OH, COOH, CONH₂、NH₂And an alkyl group substituted with a nitrogen-containing heterocycle as a substituent of a terminal group, wherein one or more CH in the alkyl group₂Part being optionally selected from-NH-, -CO-, -SO₂-and-SO-divalent radical substitution; more preferably R₁₁Is selected from

、

、

、

And

；

、-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

Forming an N-containing heterocycle;

R^ais hydrogen or alkyl;

wherein the linker moiety is selected from a bond, an optionally substituted alkyl moiety, an optionally substituted heterocyclic 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 biological material, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that interacts covalently or non-covalently with other molecules, a photocaged moiety, an actinic radiation-excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analog, a heavy atom incorporating moiety, a chemically cleavable group, a photolyzable group, a redox 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 bioactive agent, a detectable label, or a combination thereof.

Technical solution 8

The Btk affinity probe of claim 7, wherein the linker moiety is a bond.

Technical solution 9

The Btk affinity probe of claim 7, wherein the reporter moiety is a fluorophore.

Technical means 10

The Btk affinity probe of claim 9, wherein the fluorophore is a Bodipy fluorophore.

Technical means 11

The Btk affinity probe of claim 10, wherein the Bodipy fluorophore is a Bodipy FL fluorophore.

Technical means 12

Btk affinity probes having the following structure

。

Technical means 13

A method for assessing the efficacy of a potential Btk inhibitor in a mammal comprising administering a potential Btk inhibitor to said mammal, administering a Btk affinity probe of any of claims 7-12 to said mammal or a cell isolated from said 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 means 14

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

Technical means 15

A method for in vitro labeling of Btk enzyme comprising contacting a cell or tissue expressing Btk enzyme with the Btk affinity probe of claims 7-12.

Technical solution 16

A method of detecting a labeled Btk enzyme comprising separating proteins by electrophoresis, wherein the proteins comprise the Btk enzyme labeled with the Btk affinity probe of claims 7-12; 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 methods, protocols, cell lines, constructs, and reagents described herein and may vary in some embodiments. 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 references unless the context clearly dictates 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 described in the publications.

Unless otherwise specified, the term "alkyl" by itself or as part of another molecule means a straight or branched chain or cyclic hydrocarbon group or a combination thereof. In some embodiments, the alkyl chain is fully saturated, mono-unsaturated, or poly-unsaturated. In other embodiments, alkyl chains include those having the specified number of carbon atoms (i.e., C)₀-C₁₀Or C₀-₁₀ Means 0 to 10 carbons, and C₀Alkyl refers to a bond) divalent and polyvalent groups. In other embodiments, examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butylA group, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl; for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl is alkyl having one or more double or triple bonds. In another embodiment, examples of unsaturated alkyl groups include, but are not limited to, ethenyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and higher homologs and isomers. In other embodiments, unless otherwise specified, the term "alkyl" also includes derivatives of alkyl groups defined in more detail herein, such as "heteroalkyl", "haloalkyl", and "homoalkyl (homoalkyl)".

The term "biophysical probe" as used herein refers to a probe that detects or monitors a structural change in a molecule. In some embodiments, such molecules include, but are not limited to, proteins, and the "biophysical probes" are used to detect or monitor the 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 compound containing a substituent selected from the group consisting of-C (O) -, -S (O)₂-and-c(s) -including but not limited to groups containing at 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. These carbonyl groups include ketones, aldehydes, carboxylic acids, esters, and thioesters. In some embodiments, these groups are part of a linear, branched, or cyclic molecule.

The term "chemiluminescent group" as used herein refers to a group that emits light as a result of a chemical reaction without additional heating. By way of example only, luminol (5-amino-2, 3-dihydro-1, 4-phthalazinedione) is reacted with an oxidizing agent, such as hydrogen peroxide (H) in the presence of a base and a metal catalyst₂O₂) Reaction to produce the excited product (3-aminophthalate, 3-APA).

The term "chromophore" as used herein refers to a molecule that absorbs light at visible, UV or IR wavelengths.

The term "Cys 481" as used herein refers to the cysteine (i.e., "C" highlighted in bold) found in fig. 5 at the position in the kinase corresponding to Cys481 in Btk.

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

The term "dye" as used herein refers to a soluble coloring substance containing a chromophore.

The term "electron-dense group" as used herein refers to a group that scatters electrons when irradiated with an electron beam. These groups include, but are not limited to, ammonium molybdate, bismuth subnitrate, cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate, hexamethylenetetramine, 98.5%, anhydrous indium trichloride, 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 tetraphenylporphyrin (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 that 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 fluorescent donor molecule is non-radiatively transferred to an unexcited acceptor molecule, which then emits the complimentary energy at longer wavelength fluorescence.

The term "enhance" means to increase or prolong the efficacy or duration of a desired effect. For example, "enhancing" the effect of a therapeutic agent refers to the ability to increase or prolong the efficacy or duration, effect, of the therapeutic agent during treatment of a disease, disorder, or condition. As used herein, "potentiating effective amount" refers to an amount sufficient to potentiate the effect of a therapeutic agent during treatment of a disease, disorder, or condition. When used in a patient, an amount effective for this use will depend on the severity and course of the disease, disorder or condition, previous treatments, the patient's health status and response to the drug, and the judgment of the attending physician.

The term "fluorophore" as used herein refers to a molecule that upon excitation emits a photon and thereby fluoresces.

In some embodiments, the term "label" as used herein refers to a substance that is incorporated into a compound and is readily detectable, thereby detecting and/or monitoring its physical distribution.

The term "linkage" as used herein refers to a bond or chemical moiety formed by a chemical reaction between a functional group of a linker and another molecule. In some embodiments, the linkages include, but are not limited to, covalent and non-covalent linkages, and the chemical moieties include, but are not limited to, ester, carbonate, imine, phosphate, hydrazone, acetal, orthoester, peptide linkage, and oligonucleotide linkage. A hydrolytically stable linkage means that the linkage is substantially stable in water and does not react with water for long periods (perhaps even indefinitely) at applicable pH values, including but not limited to under physiological conditions. Hydrolytically unstable or degradable linkages means that the linkages can degrade in water or aqueous solutions, including, for example, blood. In other embodiments, an enzymatically labile or degradable linkage means that the linkage can be degraded by one or more enzymes. By way of example only, PEG and related polymers include degradable linkages in the polymer backbone or in linker groups between the polymer backbone and one or more terminal functional groups of the polymer molecule. These degradable linkages include, but are not limited to, ester linkages formed by the reaction of a PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on a bioactive agent, where these ester groups are typically hydrolyzed under physiological conditions to release the bioactive agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages formed by the reaction of an amine with an aldehyde; a phosphate ester bond formed by the reaction of an alcohol with a phosphate ester group; a hydrazone bond as the reaction product of a hydrazide and an aldehyde; acetal linkages as the reaction product of an aldehyde with an alcohol; ortho ester linkages as the reaction product of formate and alcohol; a peptide bond formed by an amino group (including but not limited to on the terminus of a polymer such as PEG) and a carboxyl group of a peptide; and oligonucleotide linkages formed from phosphoramidite groups (including but not limited to on the end of the polymer) and the 5' hydroxyl group of the oligonucleotide.

The phrase "measuring the activity of a reporter moiety" (or phrases of similar wording) refers to a method for quantifying (in absolute, approximate or relative terms) a reporter moiety in a system under study. In some embodiments, such methods include any method of quantifying a reporter moiety, which is a dye; a photocrosslinker; a cytotoxic compound; a drug; an affinity tag; an photoaffinity label; a reactive compound; an antibody or antibody fragment; a biological material; nanoparticles; a spin label; a fluorophore; a metal-containing moiety; a radioactive moiety; a novel functional group; groups that interact covalently or non-covalently with other molecules; a light cage portion; an actinic radiation-labile moiety; a ligand; a photoisomerizable moiety; biotin; a biotin analogue; a heavy atom-incorporating moiety; a chemically cleavable group; a photolyzable group; a redox agent; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label, or any combination of the foregoing.

The term "heavy atom-doped moiety" as used herein refers to a group doped with ions of atoms generally heavier than carbon. In some embodiments, these 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 having a particle size between about 500nm to about 1 nm.

The term "pharmaceutically acceptable" as used herein refers to a substance that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, including, but not limited to, salts, carriers, or diluents. In one embodiment, the substance is administered to an individual without causing unwanted 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 having a group that forms a bond with a molecule for which the label has an affinity after exposure to light. By way of example only, in some embodiments, such linkage is covalent or non-covalent.

The term "photocaged moiety" as used herein refers to a group that covalently or non-covalently binds other ions or molecules upon irradiation at a specific wavelength.

The term "photoisomerizable moiety" as used herein refers to a group in which one isomeric form changes to another isomeric form upon irradiation with light.

The term "radioactive moiety" as used herein refers to a group whose nucleus spontaneously emits nuclear radiation (such as alpha, beta or gamma particles); wherein the alpha particles are helium nuclei, the beta particles are electrons, and the gamma particles are high-energy photons.

The term "spin label" as used herein refers to a molecule containing an atom or group of atoms (i.e., a stable paramagnetic group) that exhibits unpaired electron spins, which in some embodiments are detected by electron spin resonance spectroscopy and in other embodiments are linked to another molecule. These spin labeling molecules include, but are not limited to, nitroxyl and nitroxide radicals (nitroxides), and in some embodiments are single spin labels or dual spin labels.

The phrase "therapeutically effective amount" of a compound of the present invention refers to an amount of the compound sufficient to treat the condition at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention is within the scope of sound medical judgment of the attending physician. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the condition being treated and the severity of the condition; the activity of the particular compound used; the specific composition used; the age, weight, general health, sex, and diet of the patient; time of administration, route of administration and rate of excretion of the particular compound used; the duration of the treatment; drugs used in combination or concomitantly with the specific compound used; and similar factors well known in the medical arts. For example, it is within the ability of one skilled in the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved.

The term "pharmaceutically acceptable carrier" as used herein refers to a non-toxic inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials that can be used 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 gum 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 as 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; ethanol 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 that is the subject 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 "substituent" (also referred to as "non-interfering substituent") refers toA group for replacing another group on the molecule. These groups include, but are not limited to, halogen, C₁-C₁₀Alkyl radical, C₂-C₁₀Alkenyl radical, C₂-C₁₀Alkynyl, C₁-C₁₀Alkoxy radical, C₅-C₁₂Aralkyl radical, C₃-C₁₂Cycloalkyl radical, C₄-C₁₂Cycloalkenyl, phenyl, substituted phenyl, tolyl, xylyl, biphenyl, C₂-C₁₂Alkoxyalkyl group, C₅-C₁₂Alkoxyaryl radical, C₅-C₁₂Aryloxyalkyl radical, C₇-C₁₂Oxyaryl radical, C₁-C₆Alkylsulfinyl radical, C₁-C₁₀Alkylsulfonyl, - (CH)₂)_m-O-(C₁-C₁₀Alkyl) wherein m is 1-8, aryl, substituted alkoxy, fluoroalkyl, heterocyclyl, substituted heterocyclyl, nitroalkyl, -NO₂, -CN, -NRC(O)-(C₁-C₁₀Alkyl), -C (O) - (C)₁-C₁₀Alkyl group), C₂-C₁₀Alkylthio alkyl, -C (O) O- (C)₁-C₁₀Alkyl), -OH, -SO₂、═S、-COOH、-NR₂Carbonyl, -C (O) - (C)₁-C₁₀Alkyl) -CF₃、-C(O)-CF₃、-C(O)NR₂、-(C₁-C₁₀Aryl) -S- (C₆-C₁₀Aryl), -C (O) - (C)₆-C₁₀Aryl), - (CH)₂)m-O-(CH₂)m-O-(C₁-C₁₀Alkyl) in which each m is 1-8, -C (O) NR₂、-C(S)NR₂、-SO₂NR₂、-NRC(O)NR₂、-NRC(S)NR₂Salts thereof, and the like. In some embodiments, each R group in the foregoing list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkaryl. When a substituent is specified by its conventional formula written from left to right, it also encompasses chemically identical substituents that may result from writing a structure from right to left, e.g., -CH₂O-is equivalent to-OCH₂-。

In some embodiments, to name onlyFor example, substituents for alkyl and heteroalkyl (including those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) 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 and-NO₂. In other embodiments, each R group in the foregoing 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 (thioalkoxy), or aralkyl. In some embodiments, when two R groups are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. In other embodiments, for example, -NR₂Including but not limited to 1-pyrrolidinyl and 4-morpholinyl.

In other embodiments, for example, substituents for aryl and heteroaryl 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)₂Fluorine (C)₁-C₄) Alkoxy and fluorine (C)₁-C₄) Alkyl groups, in a number from 0 to the total number of open valences on the aromatic ring system. In another embodiment, each R group in the above list includes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl and heteroaryl.

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

The compounds presented herein include isotopically-labeled compounds, which are identical to those presented herein for depicted in the various formulae and structures, 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 can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, such as²H、³H、¹³C、¹⁴C、¹⁵N、¹⁸O、¹⁷O、³⁵S、¹⁸F、³⁶And (4) Cl. Certain isotopically-labeled compounds described herein (e.g., such as³H and¹⁴those compounds into which the radioisotope of C is incorporated) may be used for drug and/or substrate tissue distribution detection. In addition, in other embodiments, a compound such as deuterium (i.e., a compound such as deuterium) is used²H) The isotopic substitution of (a) provides certain therapeutic advantages resulting from higher metabolic stability, such as increased in vivo half-life or reduced dosage requirements.

In other embodiments, the compounds described herein have asymmetric carbon atoms and thus exist in enantiomeric or diastereomeric forms. In some embodiments, diastereomeric mixtures are separated into their individual diastereomers, for example, by chromatography and/or fractional crystallization, based on their physicochemical differences. 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., an 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 to be part of the compositions described herein.

In additional or other embodiments, the compounds described herein are used in prodrug form. In additional or other embodiments, the compounds described herein are metabolized upon administration to an organism in need thereof to generate metabolites that are subsequently 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 referred to as polymorphs), or pharmaceutically acceptable salts. In certain embodiments, the compounds described herein exist in tautomeric forms. All tautomers are included within the scope of the compounds presented herein. In some embodiments, the compounds described herein exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. Solvated forms of the compounds set forth herein are also considered disclosed herein.

In other embodiments are compounds described herein, which exist in several tautomeric forms. All of these tautomeric forms are considered part of the compositions described herein. Likewise, for example, in some embodiments, all enol-keto forms of any compound herein are considered part of the compositions described herein.

In other embodiments, the compounds described herein are acidic and in some embodiments form salts with pharmaceutically acceptable cations. Some of the compounds herein are basic and in some embodiments form salts with pharmaceutically acceptable anions. All such salts, including disalts, are within the scope of the compositions described herein and are prepared by conventional methods in some embodiments. For example, in other embodiments, the salt is prepared by contacting an acidic entity with a basic entity in an aqueous, non-aqueous, or partially aqueous medium. Recovering the salt by using at least one of the following techniques: filtering; precipitation with a non-solvent followed by filtration; evaporating the solvent; or (in the case of aqueous solutions) lyophilized.

In some embodiments, pharmaceutically acceptable salts of the compounds disclosed herein are formed when an acidic proton present in the parent compound is replaced with a metal ion (e.g., an alkali metal ion, alkaline earth metal ion, or aluminum ion), or is coordinated to an organic base. Additionally, in other embodiments, salts of the starting materials or intermediates are used to prepare salt forms of the disclosed compounds.

In other embodiments, types of pharmaceutically acceptable salts include, but are not limited to: (1) acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or 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' -methylenebis- (3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, cinnamic acid, mandelic acid, methanesulfonic acid, succinic acid, tartaric acid, cinnamic acid, mandelic acid, methanesulfonic acid, succinic acid, tartaric acid, citric acid, tartaric acid, cinnamic acid, tartaric acid, and mixtures of any of the compounds of any of the compounds of any of the compounds of the, Stearic acid, muconic acid, and the like; (2) when the acidic proton present in the parent compound is replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion); or a salt formed when coordinated to an organic base. In yet another 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 salt are analyzed and identified using a variety of 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 will be appreciated that, in some embodiments, reference to a salt includes a solvent addition form or a crystal form thereof, particularly a solvate or polymorph. In other embodiments, the solvate contains a stoichiometric or non-stoichiometric amount of solvent, and is typically formed with a pharmaceutically acceptable solvent (such as water, ethanol, etc.) during crystallization. In yet another embodiment, hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Polymorphs include different crystal packing arrangements of compounds of the same elemental composition. In one embodiment, the polymorphs have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shape, optical and electrical properties, stability and solubility. In other embodiments, various factors such as recrystallization solvent, crystallization rate, and storage temperature cause a single crystal form to dominate.

In other embodiments, the screening and characterization of 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 adsorption, and microscopy. In yet another embodiment, the thermal analysis methods focus on thermochemical degradation or thermophysical processes (including but not limited to polymorphic transformations), and these methods are used to analyze the relationship between polymorphs, to determine weight loss to find glass transition temperature, or for excipient compatibility studies. These methods include, but are not limited to, Differential Scanning Calorimetry (DSC), Modulated Differential Scanning Calorimetry (MDSC), thermogravimetric analysis (TGA), and thermogravimetric 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). 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 (in a gas or water vapor atmosphere) with EDX, IR microscopy, and raman microscopy.

Drawings

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

fig. 1 shows the structure of a representative Btk inhibitor.

FIG. 2 shows the structure of a representative fluorescent probe.

FIG. 3 shows the components of an affinity probe.

Figure 4 shows a general scheme for measuring target binding by competition assays between inhibitors and affinity probes.

Figure 5 shows an alignment of the ATP-binding pocket in the kinase containing a conserved cysteine (arrow) corresponding to Cys481 in Btk. Protein accession numbers are also shown.

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

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

Fig. 8 shows the endogenous Btk labeled mainly by probe 14 in living cells (course of concentration).

FIG. 9 shows the results of time course experiments for cell labeling.

Fig. 10 shows the results of Btk immunoprecipitation from probe 14 labeled lysates. Lane 1: a cell lysate; lane 2: supernatant after removal of intrinsic IgG; lane 3: the supernatant after immunoprecipitation; lane 4: supernatant of the last washing before elution; lane 5: the first eluted supernatant was prepared by applying LDS sample buffer to protein a sepharose beads.

FIG. 11 shows that the labeling of Btk by probe 14(0.5 μ M) was completely competed by ibrutinib and compound 2(1 μ M).

Fig. 12 shows measurement of extent of Btk occupancy by inhibitors (ibrutinib and compound 2) in living cells. The band density was measured by Gelpro32 and IC50 values were determined using Graphpad Prism.

FIG. 13 shows competition experiments in OCI-Ly7 cells (a) and Jurkat cells (b). Mixing 1.5X 10⁶The cells were pre-incubated with compound for 1h at 1. mu.M, then labeled with probe 14 for 2h at 0.5. mu.M, then lysed, quantified and analyzed by SDS/PAGE and fluorescence gel scanning (fluorescence, CY 2).

Detailed Description

Disclosed herein is the synthesis and characterization of Btk inhibitors. Also disclosed herein is the synthesis and characterization of cell permeable probes that label Btk at unique non-catalytic cysteine residues in the ATP-binding pocket.

Btk inhibitors

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

、

And

；

、

、

、

、

、

、

And

(ii) a More preferably R₁Is selected from

、

、

、

、

And

；

wherein

p is 0, 1,2 or 3, preferably 0;

q is 0, 1,2 or 3, preferably 0;

R₁₁is selected from OH, COOH, CONH₂、NH₂And a substituent of a terminal group of the nitrogen-containing heterocycle; preferably R₁₁Is selected from OH, COOH, CONH₂、NH₂And an alkyl group substituted with a nitrogen-containing heterocycle as a substituent of a terminal group, wherein one or more CH in the alkyl group₂The radicals being optionally selected from-NH-, -CO-, -SO₂-and-SO-divalent radical substitution; more preferably R₁₁Is selected from

、

、

、

And

。

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

btk active probe compounds

The Btk affinity probe compounds described herein consist of a moiety comprising a Btk inhibitor (or 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 other 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 another embodiment, the Btk affinity probe forms a non-covalent bond with at least one non-catalytic residue of Btk. In other embodiments, the Btk affinity probe forms a hydrogen bond within the ATP-binding pocket of Btk. In yet another embodiment, the Btk affinity probe has van der waals attraction to the Btk enzyme.

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

In some embodiments, the Btk affinity probe is cell permeable.

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

One aspect is a Btk affinity probe of formula (Ic) as follows:

wherein the Btk inhibitor moiety can be derived from a compound having the general formula (Ia) or (Ib-1) or (Id) described above;

wherein X and Y are independently selected from the group consisting of a bond,

Wherein

Forming an N-containing heterocycle;

R^ais hydrogen or alkyl.

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

More preferably R₁Is selected from

Wherein

p is 0, 1,2 or 3, preferably 0;

q is 0, 1,2 or 3, preferably 0;

。

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

in one embodiment, the Btk inhibitor moiety is selected from

In one embodiment, the Btk inhibitor is derived from an irreversible inhibitor of Btk. In some embodiments, these irreversible inhibitors of Btk should have at least one of the following characteristics: potency, selectivity and cell permeability. In other embodiments, these irreversible inhibitors of Btk have at least two, and in other embodiments, at least all, of the above-described characteristics.

In another embodiment, the linker moiety is selected from the group consisting of a bond, a polymer, a water-soluble polymer, an optionally substituted alkyl, an optionally substituted heteroalkyl, an optionally substituted heterocycloalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl alkyl, an optionally substituted heterocycloalkyl alkenyl, a substituted heterocycloalkyl alkyl, a substituted cycloalkyl, a substituted heterocycloalkyl, a substituted cycloalkyl, a substituted or a substituted cycloalkyl, a substituted or a substituted cycloalkyl, a substituted or a substituted cycloalkyl, a substituted or a substituted cycloalkyl, a substituted or a substituted cycloalkyl, a substituted or a,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 the group consisting of aziridine, oxetane, thietane, oxetane, pyrroline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, pyrazole, pyrrole, imidazole, triazole, tetrazole, oxazole, isoxazole, ethylene oxide, thiazole, isothiazole, dithiolane, furan, thiophene, piperidine, tetrahydropyran, thiacyclohexane, pyridine, pyran, thiopyran, pyridazine, pyrimidine, pyrazine, piperazine, oxazine, thiazine, dithiane, and dioxane. In some embodiments, the heterocycle is piperazine. In other embodiments, the linker moiety is substituted with halogen, CN, OH, NO₂Alkyl, S (O) and S (O)₂Optionally substituted. 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 other embodiments, the linker moiety is stable. In yet another embodiment, the linker moiety does not substantially affect the reaction of the reporter moiety. In other embodiments, the linker moiety provides chemical stability to the Btk affinity probe. In other embodiments, the linker moiety provides sufficient solubility to the Btk affinity probe.

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

In some embodiments, examples of hydrophilic polymers include, but are not limited to: polyalkyl ethers and alkoxy-terminated analogs thereof (e.g., polyoxyethylene glycol, polyoxyethylene/propylene glycol and methoxy-or ethoxy-terminated analogs thereof, polyoxyethylene glycol, the latter also known as polyethylene glycol or PEG); polyvinylpyrrolidone; polyvinyl alkyl ethers; polyoxazolines, polyalkyloxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkylacrylamides and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivatives thereof); polyhydroxyalkyl acrylate; polysialic acid and analogs thereof, hydrophilic peptide sequences; polysaccharides and derivatives thereof, including dextran and dextran derivatives, e.g., carboxymethyl dextran, dextran sulfate, aminodextran; cellulose and its derivatives, for example, carboxymethyl cellulose, hydroxyalkyl cellulose; chitin and its derivatives, such as chitosan, succinyl chitosan, carboxymethyl chitin, carboxymethyl chitosan; hyaluronic acid and derivatives thereof; starch; an alginate; chondroitin sulfate; albumin; amylopectin and carboxymethyl amylopectin; polyamino acids and derivatives thereof, for example, polyglutamic acid, polylysine, polyaspartic acid, polyaspartamide; maleic anhydride copolymers such as: styrene maleic anhydride copolymer, divinyl ethyl ether maleic anhydride copolymer; polyvinyl alcohol; copolymers thereof, terpolymers thereof, mixtures thereof and derivatives of the foregoing. In other embodiments, the water-soluble polymer is in any structural form, including, but not limited to, linear, forked, or branched. In some embodiments, a water-soluble polymer backbone having from 2 to about 300 ends is particularly useful. In other embodiments, polyfunctional polymer derivatives include, but are not limited to, linear polymers having two ends, wherein each end is bonded to the same or different functional groups. In some embodiments, the water soluble polymer comprises a poly (ethylene glycol) moiety. In other embodiments, the molecular weight of the polymer is in a broad range, including, but not limited to, about 100 Da to about 100,000 Da or greater. In other embodiments, the polymer has a molecular weight of about 100 Da to 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 about 100 Da to 50,000 Da. In some embodiments, the molecular weight of the polymer is about 100 Da to 40,000 Da. In some embodiments, the molecular weight of the polymer is about 1,000 Da to 40,000 Da. In some embodiments, the molecular weight of the polymer is about 5,000 Da to 40,000 Da. In some embodiments, the molecular weight of the polymer is about 10,000 Da to 40,000 Da. In some embodiments, the poly (ethylene glycol) molecule is a branched polymer. In other embodiments, the branched PEG has a molecular weight of about 1,000 Da to 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 PEG is about 1,000 Da to about 50,000 Da. In some embodiments, the molecular weight of the branched PEG is about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the branched PEG is about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the branched PEG is about 5,000 Da to about 20,000 Da. The above list of substantially water-soluble backbones is by no means exhaustive and merely illustrative, and in some embodiments polymeric materials having the properties as described above are suitable for use in the methods and compositions described herein.

In other embodiments, the number of water-soluble polymers attached to the Btk inhibitor moiety and reporter moiety described herein are adjusted to provide altered (including, but not limited to, increased or decreased) pharmacological, pharmacokinetic or pharmacodynamic characteristics such as in vivo half-life. In some embodiments, the half-life of the Btk affinity probe is increased by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, to about 2-fold, about 5-fold, about 10-fold, about 50-fold, or at least about 100-fold as compared to the Btk affinity probe without the 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

Forming an N-containing heterocyclic ring. In one embodiment, X is NR^a(CO). In another embodiment, X is a bond. In another embodiment, X is-O (CO) -.

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

Wherein

Forming an N-containing heterocyclic ring. In yet another embodiment, Y is a bond. In one embodiment, Y is-NR^a(CO) -. In yet another embodiment, R^aIs hydrogen. In yet another embodiment, R^aIs an alkyl group.

In another 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 biological material, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that interacts covalently or non-covalently with other molecules, a photocage moiety, an actinic radiation-excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analog, a heavy atom incorporating moiety, a chemically cleavable group, a photolyzable group, a redox 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 bioactive agent, a detectable label, or a combination thereof.

In another embodiment, the reporter moiety is a fluorophore. In another 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-bis (2,4, 6-trimethylphenyl) -3,4:9, 10-perylene bis (dicarboximide), HPTS, ethyl eosin, DY-490XL MegaStokes, DY 485-XL MegaStokes, Adirondack Green 520, ATTO 465, ATTO 488, ATTO 495, YOYO-1,5-FAM, BCECF, dichlorofluorescein, rhodamine 110, rhodamine 123, rhodamine Green, YO-PRO-1, SYX Green, SodiBR, SYDEX Green, SYEx Green, AluFlua-Fluo-3, FluoFluoC-3, FluoC-1, FluoFluoC-1, FluoC, Fluoro-emerald, Yoyo-1 ssDNA, Yoyo-1 dsDNA, Yoyo-1, SYTO RNASelect, Diversa Green-FP, Dragon Green, EvaGreen, surfGreen 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), FlaSH-CCXXCC, Azami Green monomeric, Aza Green, EGFP (Campbell Tsen 2003), EGFP (Patterson 2001), fluorescein, Kaede Green, 7-benzylamino-4-nitrophenyl-2-oxa-1, 3-diazole, Bexmil, Aminomycin, and Glo.

In another 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 another embodiment, the fluorophore is BODIPY FL. In certain embodiments, the fluorophore is not BODIPY 530. In some embodiments, the fluorophore has an excitation maximum of about 500 to about 600 nm. In some other embodiments, the fluorophore has an excitation maximum of about 500 to about 550 nm. In other embodiments, the fluorophore has an excitation maximum of about 550 to about 600 nm. In yet another embodiment, the fluorophore has a maximum excitation of about 525 to about 575 nm. In other embodiments, the fluorophore has an excitation maximum of about 510 to about 670 nm. In another embodiment, the fluorophore has an excitation maximum of about 510 to about 600 nm. In another embodiment, the fluorophore has an excitation maximum of about 600 to about 670 nm. In another embodiment, the fluorophore has an excitation maximum of about 575 to about 625 nm.

In some embodiments, the formed linkage is a stable linkage. In other embodiments, where the conjugate comprises two moieties, 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 a means to control and determine the distance between the Btk inhibitor moiety and the reporter moiety. In addition, in some embodiments, the linker moiety is selected so as to preserve the solubility of the probe. In other embodiments, the number and order of units comprising the linker moiety is selected so as to control the length between the first and second component moieties and the hydrophobic and hydrophilic characteristics of the linker.

In the context of the present invention, spatial separation refers to thermochemically and photochemically inactive manufacturing-distance groups and in some embodiments is used to link two or more different moieties of the type defined above. In other embodiments, the spacer is selected based on various characteristics including its hydrophobicity, hydrophilicity, molecular flexibility, and length. Thus, in some embodiments, the spacer comprises a chain of carbon atoms optionally interrupted or terminated by one or more heteroatoms such as oxygen atoms, nitrogen atoms and/or sulfur atoms. Thus, in some embodiments, the spacer comprises one or more amide, ester, amino, ether and/or thioether functional groups, and optionally an aromatic or mono/polyunsaturated hydrocarbon, a polyoxyethylene such as polyethylene glycol, an oligo/polyamide such as poly-. alpha-alanine, polyglycine, polylysine and peptides, oligosaccharides, oligo/polyphosphates in general. Furthermore, in other embodiments, the spacer consists of a merged unit thereof. In other embodiments, the length of the spacer can be varied to account for the desired or necessary positioning and spatial orientation of the active/functional moieties of the Btk affinity probes.

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

In some embodiments, compound (14) retains the solubility and membrane permeability of compound (11) to allow detection and quantification 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 not limited to affinity chromatography; anion exchange or cation exchange chromatography (using, including but not limited to, DEAE SEPHAROSE); silica gel chromatography; reversed phase HPLC; gel filtration (using, including but not limited to, SEPHADEX G-75); hydrophobic interaction chromatography; size exclusion chromatography, metal chelate chromatography; ultrafiltration/diafiltration (diafiltration); ethanol precipitation; ammonium sulfate precipitation; focusing chromatography; displacement chromatography; electrophoresis (including but not limited to preparative isoelectric focusing), differential solubilization (including but not limited to ammonium sulfate precipitation), or extraction. In other embodiments, the apparent molecular weight is estimated by GPC by comparison with globulin standards (PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989, 293-306).

Further, in some embodiments, the synthetic procedures disclosed below include various purifications, such as column chromatography, flash chromatography, Thin Layer Chromatography (TLC), recrystallization, distillation, High Pressure Liquid Chromatography (HPLC), and the like. Additionally, in other embodiments, various techniques for identifying and quantifying chemical reaction products are also used, such as proton and carbon-13 nuclear magnetic resonance: (¹H and¹³c NMR), infrared and ultraviolet spectra (IR and UV), X-ray crystallography, Elemental Analysis (EA), HPLC, and Mass Spectrometry (MS).

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

The abbreviations used herein have the following meanings

DMF	Dimethyl formamide
		HOBt	Hydroxybenzotriazoles
EDCI	1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
		DCM	Methylene dichloride
TFA	Trifluoroacetic acid
		THF	Tetrahydrofuran (THF)
DIEA	N, N-diisopropylethylamine
		EtOAc	Ethyl acetate
MeOD	Deuterated methanol
		HATU	1- [ bis (dimethylamino) methylene]-1H-1,2, 3-triazolo [4,5-b]Pyridinium 3-oxide hexafluorophosphate

Synthetic examples

Unless otherwise indicated, all reagents were purchased commercially and used without further purification. All yields refer to chromatographic yields. Anhydrous Dimethylformamide (DMF) was distilled from calcium hydride. Brine refers to a saturated solution of sodium chloride in distilled water. The reaction was monitored by Thin Layer Chromatography (TLC) performed on a 0.25mm bench silica gel plate (HSGF254) using a UV lamp as a chromogenic agent. Flash column chromatography was performed using silica gel on a bench scale (ZCX-II, particle size 0.048-0.075 mm). 1H-NMR and 13C-NMR spectra were recorded at Bruker Advance 400 (1H: 400 MHz, 13C: 100 MHz) or Bruker Advance 300 (¹H: 300 MHz, ¹³C: 75 MHz) was recorded on a spectrometer at ambient temperature, and the chemical shift values were recorded relative to TMS (delta) as a standard_H0.00 and delta_C0.00), dimethyl sulfoxide (. delta.))_H2.50 and delta_C39.52) or methanol (. delta.)_H3.31 and δ_C49.00) in ppm. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constant and number of protons. HR-MS was obtained using Bruker Apex IV RTMS. The purity of the compounds was determined by HPLC chromatograms obtained on Agilent 1200 HPLC or 1260 HPLC. Analysis was performed by an Agilent PN959990-902 Eclipse Plus C18250 mm X4.6 mm column using a water-MeCN gradient from 50% to 98% or 50% to 65% MeCN within 10 min. Detection at 254 nm, using the average peakArea to determine purity. All compounds are defined as>95% pure.

EXAMPLE 1 Synthesis of N- (2- (3- (3-acrylamidopropionamido) phenylamino) pyrimidin-5-yl) -2-methyl-5- (3- (trifluoromethyl) benzamido) benzamide (3)

A procedure similar to example 4 was repeated except for replacing N- (tert-butoxycarbonyl) -L-leucine with 3- (tert-butoxycarbonylamino) propionic acid in step 1 of example 4 to give the title compound as a white solid.

HRMS-ESI C₃₂H₂₈F₃N₇NaO₄ [M+Na⁺]The calculated value of (a): 654.2053, respectively; measured value: 654.2058.

example 2 Synthesis of (S) -1-acryloyl-N- (3- ((5- (2-methyl-5- (3- (trifluoromethyl) benzamido) pyrimidin-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 of example 4 to give the title compound as a white solid.

HRMS-ESI C₃₄H₃₀F₃N₇NaO₄ [M+Na⁺]The calculated value of (a): 680.2209, respectively; measured value: 680.2204。

Example 3 Synthesis of (S) -N- (2- ((3- (2-acrylamidopropionamido) 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) propionic acid in step 1 of example 4 to give the title compound as a white solid.

EXAMPLE 4 Synthesis of (S) -N- (2- ((3- (2-acrylamido-4-methylpentanamido) phenyl) amino) pyrimidin-5-yl) -2-methyl-5- (3- (trifluoromethyl) benzamido) benzamide (6)

Step 1:

N- (2- ((3-aminophenyl) amino) pyrimidin-5-yl) -2-methyl-5- (3- (trifluoromethyl) benzamido) benzamide (intermediate 1) (0.101g, 0.2mmol) was dissolved in 2ml dry DMF, cooled to 0 ℃ and Et was added at 0 ℃₃N (0.14ml, 1mmol), HOBt (0.032g, 0.24mmol), N- (tert-butoxycarbonyl) -L-leucine (0.069g, 0.3mmol) and slow EDCI (0.077g, 0.4 mmol). The reaction was allowed to slowly reach room temperature and allowed to react overnight. After the reaction was stopped, 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 purifiedPurification by column chromatography (gradient: 30-50% EtOAc/hexane) afforded tert-butyl (S) -4-methyl-1- (3- (5- (2-methyl-5- (3- (trifluoromethyl) benzamido) pyrimidin-2-ylamino) phenylamino) -1-oxopent-2-ylcarbamate (0.105g) as a pale yellow solid.

Step 2:

Tert-butyl (S) -4-methyl-1- (3- (5- (2-methyl-5- (3- (trifluoromethyl) benzamido) pyrimidin-2-ylamino) phenylamino) -1-oxopent-2-ylcarbamate (0.105g, 0.15mmol) was dissolved in 6ml DCM and treated with 3ml TFA. The mixture was stirred at room temperature for 2h and concentrated in vacuo. The crude product (about 0.17mmol) was dissolved in 2ml THF, cooled to 0 ℃, DIEA (56 µ L,0.34mmol), 1ml water was added, followed by slow addition of acryloyl chloride (21 µ L, 0.25 mmol). The reaction was allowed to reach room temperature for 2 hours. After the reaction was stopped, 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/hexanes) to give (S) -N- (2- ((3- (2-acrylamido-4-methylpentamamido) phenyl) amino) pyrimidin-5-yl) -2-methyl-5- (3- (trifluoromethyl) benzamido) benzamide (6) (40.8mg, 30% over three steps) as a white solid.

HRMS-ESI C₃₅H₃₄F₃N₇NaO₄ [M+Na⁺]The calculated value of (a): 696.2522, respectively; measured value: 696.2516.

example 5 Synthesis of (S) -N- (2- ((3- (2-acrylamido-3-phenylpropionamido) phenyl) amino) pyrimidin-5-yl) -2-methyl-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-phenylpropionic acid in step 1 of example 4 to give the title compound as a white solid.

HRMS-ESI C₃₈H₃₂F₃N₇NaO₄ [M+Na⁺]The calculated value of (a): 730.2366, respectively; measured value: 730.2366.

example 6 Synthesis of (S) -N- (2- ((3- (2-acrylamido-3-hydroxypropionamido) 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-hydroxypropionic acid in step 1 of example 4 to give the title compound as a pale yellow solid.

HRMS-ESI C₃₂H₂₈F₃N₇NaO₅ [M+Na⁺]The calculated value of (a): 670.2002, respectively; measured value: 670.1999.

example 7 Synthesis of (S) -2-acrylamido-N1- (3- ((5- (2-methyl-5- (3- (trifluoromethyl) benzamido) pyrimidin-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 of example 4 to give the title compound as a pale yellow solid.

HRMS-ESI C₃₃H₃₀F₃N₈O₅ [M+H⁺]The calculated value of (a): 675.2291, respectively; measured value: 675.2286.

example 8 Synthesis of (S) -4-acrylamido-5- ((3- ((5- (2-methyl-5- (3- (trifluoromethyl) benzamido) pyrimidin-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) glutaric acid in step 1 of example 4 to give the title compound as a pale yellow solid.

HRMS-ESI C₃₄H₃₁F₃N₇O₆ [M+H⁺]The calculated value of (a): 690.2288, respectively; measured value: 690.2283.

EXAMPLE 9 Synthesis of (S) -N- (2- ((3- (2-acrylamido-6-aminohexanamido) phenyl) amino) pyrimidin-5-yl) -2-methyl-5- (3- (trifluoromethyl) benzamido) benzamide trifluoroacetate (11)

Step 1:

N- (2- ((3-aminophenyl) amino) pyrimidin-5-yl) -2-methyl-5- (3- (trifluoromethyl) benzamido) benzamide (intermediate 1) (0.07g, 0.14mmol), N α - [ (9H-fluoren-9-ylmethoxy) carbonyl ] -N ε - (tert-butoxycarbonyl) -L-lysine (0.101g, 0.21mmol), HATU (0.105g, 0.28mmol) were dissolved in dry DMF, cooled to 0 ℃ and DIEA (46 μ L, 0.28mmol) was added. The reaction was allowed to slow to room temperature and allowed to react overnight. While stopping the reaction, 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/hexanes) to give tert-butyl (S) - (6- ((3- ((5- (2-methyl-5- (3- (trifluoromethyl) benzamido) pyrimidin-2-yl) amino) phenyl) amino) -6-oxohexane-1, 5-diyl) dicarbamate (9H-fluoren-9-yl) methyl ester (0.128g, 96%) as a yellow solid.

Step 2:

Tert-butyl (S) - (6- ((3- ((5- (2-methyl-5- (3- (trifluoromethyl) benzamido) pyrimidin-2-yl) amino) phenyl) amino) -6-oxohexane-1, 5-diyl) dicarbamate (9H-fluoren-9-yl) methyl ester (0.128g, 0.13mmol) was dissolved in 1ml of dry DMF, treated with 1ml of morpholine, and reacted at room temperature for 3 hours. While stopping the reaction, 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/DCM) to give tert-butyl (S) -5-amino-6- (3- (5- (2-methyl-5- (3- (trifluoromethyl) benzamido) pyrimidin-2-ylamino) phenylamino) -6-oxohexylcarbamate (0.084g, 88%) as a pale yellow solid.

Step (ii) of3:

Tert-butyl (S) -5-amino-6- (3- (5- (2-methyl-5- (3- (trifluoromethyl) benzamido) pyrimidin-2-ylamino) phenylamino) -6-oxohexylcarbamate (0.084g, 0.114mmol) was dissolved in 1ml THF, cooled to 0 deg.C, DIEA (38. mu.L, 0.23mmol), 0.5ml water were added, followed by slow addition of acryloyl chloride (18. mu.L, 0.23 mmol). The reaction was allowed to reach room temperature for 2 hours. While stopping the reaction, 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 and concentrated to give the crude product as a yellow solid (0.080 g). The latter was dissolved in 1ml DCM and treated with 0.5ml TFA. The mixture was stirred at rt for 2h and concentrated in vacuo, and the resulting solid was washed with diethyl ether to give the title compound (11) as a yellow solid (0.037 g, via 2 steps 47%).

HRMS-ESI C₃₅H₃₆F₃N₈O₄ [M+H⁺]The calculated value of (a): 689.2812, respectively; measured value: 689.2807.

EXAMPLE 10 Synthesis of (S) -N- (2- ((3- (2-acrylamido-3- (1H-imidazol-5-yl) propionamido) phenyl) amino) pyrimidin-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) propionic acid in step 1 of example 4 to give the title compound as a pale yellow solid.

HRMS-ESI C₃₅H₃₁F₃N₉O₄ [M+H⁺]The calculated value of (a): 698.2451, respectively; measured value: 698.2447.

EXAMPLE 11 Synthesis of (S) -N- (2- ((3- (2-acrylamido-6- (pent-4-alkynylamido) hexanamido) phenyl) amino) pyrimidin-5-yl) -2-methyl-5- (3- (trifluoromethyl) benzamido) benzamide (13)

Similar procedure as in example 12 was repeated except for replacing BODIPY FL with pent-4-ynoic acid to prepare the title compound as a pale yellow solid.

HRMS-ESI C₄₀H₃₉F₃N₈NaO₅ [M+Na⁺]The calculated value of (a): 791.2893, respectively; an actual measurement value; 791.2889.

example 12

(S) -3- (3- (5-acrylamido-6- (3- (5- (2-methyl-5- (3- (trifluoromethyl) benzamido) 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] diazaborban-4-ium-5-ide (14)

Compound (11) (0.038g, 0.055mmol), 4-difluoro-5, 7-dimethyl-4-bora-3 a,4 a-diaza- (S) -asymmetric indacene-3-propionic acid (BODIPY FL) (0.015g, 0.05mmol), HATU (0.038g, 0.1mmol) were dissolved in dry DMF, cooled to 0 ℃ and then DIEA (41. mu.L, 0.25mmol) was added. The reaction was allowed to slow to room temperature and allowed to react overnight. While stopping the reaction, 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/hexanes-1% MeOH/EtOAc) to give the indicated product (14) (0.033g, 70%) as a red solid.

HRMS-ESI C₄₉H₄₈BF₅N₁₀NaO₅ [M+Na⁺]The calculated value of (a): 985.3720, respectively; measured value: 985.3717.

biological assay

Enzymatic assay for kinases

Kinases were purchased from Carna Biosciences. Kinase enzymatic assays were performed according to the protocol specified for the HTRF KinEase ™ assay sold by Cisbio Bioassays.

Kinetic Studies of Btk inhibitors

The kinase assay was performed at room temperature. Serial dilutions of compounds in DMSO were added to reaction buffer containing 0.5nM Btk and incubated for different periods of time (0min, 4min, 8min, 12min, 16min and 20 min). The enzymatic reaction is initiated by adding ATP and a substrate to the reaction mixture. Enzyme activity was measured using the HTRF KinEase ® assay. From the book Enzyme Kinetics of Hans Bisswanger [ Bisswanger, H.Enzyme Kinetics -principles and methods, 103-106 (Weinheim, 2002)]And (5) guiding data analysis.

Recombinant protein marker assay

0.5 μ g of recombinant Btk were incubated with increasing concentrations of probe 14 for 2h in 25 μ L of PBS buffer and then analyzed by SDS/PAGE and fluorescent gel scanning (fluorescence, CY 2). Gels were then blotted (blotted) and total Btk levels were detected by standard silver staining. The concentration course marker program is similar to the time course marker program.

Cell marker assay

Btk was labeled with probe 14. In total 1.5X 10⁶The cells were treated with 1 μ M probe 14 for various lengths of time (5 min, 10 min, 20min, 30 min, 1h, 2h, 3 h or 4 h), washed, lysed in cell lysis buffer (Beyotime) containing 1mM PMSF and 10 mM NaF (Invitrogen) and centrifuged. The sample protein concentration was quantified using a NanoDrop 2000 spectrophotometer, adjusted to the same concentration and then analyzed by SDS/PAGE and fluorescence gel scanning (fluorescence, CY 2). The gels were then analyzed by blotting and total Btk levels were detected by standard western blotting. The concentration course marker program is similar to the time course marker program.

Immunoprecipitation of Btk. LY7 cells were treated with 0.5. mu.M Probe 14 for 2h in binding buffer (20 mM Na) containing phosphatase and protease inhibitors₃PO₄pH 7.5, 150 mM NaCl). The lysates obtained were pre-incubated with Protein A Sepharose beads (GE healthcare, 17-5138-01) to remove intrinsic cellular IgG proteins. Meanwhile, rabbit anti-Btk (CST, 8574S) was preincubated with Protein A Sepharose beads for 2 hours at 4 ℃. The pretreated lysate was then added to the pretreated immobilized Protein A agarose gel and incubated at 4 ℃ for 2 hours. The immunocomplexes were washed four times with binding buffer, 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.

And (4) performing a competition test. LY7 cells were preincubated with compound (1 μ M) for 1h, followed by labeling with probe 14 under appropriate time and concentration conditions. The cells were then lysed and analyzed as described above.

Target binding of Btk inhibitors. LY7 cells were preincubated with different concentrations of compound for 1h, followed by labeling with probe 14. The samples were then lysed and analyzed as described above. The Btk band density was analyzed using Gelpro32 software to obtain half maximal active site occupancy values.

Discussion of results

Structure-activity relationships for Btk inhibitors

The Btk inhibitor activity (based on kinase enzymatic assays) of the compounds of the invention is shown in the following table:

values were determined by a single run in duplicate, and all remaining values were the average of two separate measurements.

Compound 14 is a selective affinity probe for Btk

When the recombinant Btk (0.5 μ g) was incubated with increasing concentrations of probe 14, the fluorescence signal increased accordingly, selecting 0.5 μ M as the probe concentration for the following step, as it already provided a sufficiently strong signal. When probe 14 was incubated with Btk for increasing periods of time, the brightness of the fluorescent signal reached a maximum at 2 hours (fig. 6 and 7).

In live OCI-Ly7 cells, a major band was present at the expected molecular weight of Btk (about 76 kDa) and several minor bands were present at lower molecular weights. The main band is also immunoreactive with anti-Btk antibodies. Concentration process experiments again showed that Btk band intensity was saturated at 0.5 μ M probe 14. Time course experiments also showed that an incubation time of 2 hours was sufficient for labeling (fig. 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). In summary, Btk was indeed the main band labeled by probe 14 in OCI-Ly7 cells (FIG. 13 a). As expected, no significant marker was detected in Jurkat cells (T cell line that does not express Btk) (fig. 13 b).

Inhibitors of the 2, 5-diaminopyrimidine series bind Btk directly in living cells

After optimizing the marking conditions, the probes are checkedNeedle 14 can be used to assess target binding of the inhibitor to Btk. Two types of structurally different Btk inhibitors were examined: the clinically approved Btk drug ibrutinib and compound 2 (which contains the same backbone as probe 14). Cells were first incubated with inhibitor for 1 hour, followed by 0.5 μ M probe 14 for 2 hours. As shown in fig. 11, both compounds effectively blocked labeling of Btk by probe 14 at 1 μ M. To measure the extent of Btk occupancy by the inhibitor in living cells, OCI-Ly7 cells were incubated with increasing concentrations of the compound for 1 hour, followed by labeling with probe 14 for 2 hours. After cell lysis, the protein content was loaded directly onto the gel. After electrophoresis, the fluorescence intensity of the bands was measured. IC of ibrutinib and compound 2 occupancy of Btk as presented in figure 12₅₀Values 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 in light thereof are intended 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

1. A compound having the general formula (Id):

wherein

p is 0, 1,2 or 3;

q is 0, 1,2 or 3; and is

R₁₁Is selected from

。

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein p is 0 and q is 0.

3. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

。

4. a pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

5. Use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of a disease selected from the group consisting of autoimmune diseases, heteroimmune diseases, inflammatory diseases, cancer and thromboembolic diseases.

6. Use of a compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, in the preparation of a Btk affinity probe.

7. The use of any one of the following compounds or a pharmaceutically acceptable salt thereof in the preparation of a Btk affinity probe:

。

8. a Btk affinity probe of formula (Ic) below:

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

wherein

p is 0, 1,2 or 3;

q is 0, 1,2 or 3;

R₁₁is selected from

；

Wherein X, Y and the linker moiety are independently selected from a bond; and

wherein the reporter moiety is a Bodipy fluorophore.

9. The Btk affinity probe of claim 8, wherein p is 0 and q is 0.

10. The Btk affinity probe of claim 8, wherein the Bodipy fluorophore is a Bodipy FL fluorophore.

11. A Btk affinity probe having the structure:

。

12. use of the Btk affinity probe of any one of claims 8-11 in the preparation of a reagent for assessing the efficacy of a potential Btk inhibitor in a mammal, the method comprising administering a potential Btk inhibitor to the mammal, administering the Btk affinity probe of any one of claims 8-11 to the mammal, or a cell 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.

13. Use of the Btk affinity probe of any one of claims 8-11 in the preparation of a reagent for a method of assessing the pharmacodynamics of a Btk inhibitor in a mammal, the method comprising administering the Btk inhibitor to a plurality of mammals, administering the Btk affinity probe of any one of claims 8-11 to the plurality of mammals or cells isolated from the plurality of mammals, and measuring the activity of the Btk affinity probe at different time points after administration of the inhibitor.

14. Use of the Btk affinity probe of any one of claims 8-11 in the preparation of a reagent for use in a method for in vitro labeling of a Btk enzyme, the method comprising contacting a cell or tissue expressing the Btk enzyme with the Btk affinity probe of any one of claims 8-11.