WO2017063103A1 - Nouveaux inhibiteurs et nouvelles sondes pour kinases et leurs utilisations - Google Patents

Nouveaux inhibiteurs et nouvelles sondes pour kinases et leurs utilisations Download PDF

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WO2017063103A1
WO2017063103A1 PCT/CN2015/000691 CN2015000691W WO2017063103A1 WO 2017063103 A1 WO2017063103 A1 WO 2017063103A1 CN 2015000691 W CN2015000691 W CN 2015000691W WO 2017063103 A1 WO2017063103 A1 WO 2017063103A1
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btk
group
moiety
affinity probe
compound
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PCT/CN2015/000691
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Zhengying Pan
Yingying ZUO
Xitao LI
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Peking University Shenzhen Graduate School
Beijing Reciproca Pharmaceuticals Co. Ltd.
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Priority to CN201580085270.XA priority Critical patent/CN108779078B/zh
Priority to PCT/CN2015/000691 priority patent/WO2017063103A1/fr
Publication of WO2017063103A1 publication Critical patent/WO2017063103A1/fr

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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
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    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
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    • C07D—HETEROCYCLIC COMPOUNDS
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    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom

Definitions

  • the present invention relates to 2, 5-diaminopyrimidine-based Bruton’s tyrosine kinase inhibitors.
  • the present invention also relates to 2, 5-diaminopyrimidine-based affinity probes for Bruton’s tyrosine kinase and uses of such probes for measuring the activity of Btk, for assessing the activity of modulators of Btk, and for assessing the pharmacokinetic and pharmacodynamic properties of such modulators.
  • Bruton’s tyrosine kinase is a cytosolic non-tyrosine kinase that is expressed only in hematopoietic cells, except in natural killer and T cells. Btk participates in several signaling pathways, particularly in the B cell receptor (BCR) pathway, which is crucial in B-cell development and differentiation [Mohamed, A. J. et al. Bruton′s tyrosine kinase (Btk) : function, regulation, and transformation with special emphasis on the PH domain. Immunol. Rev. 228, 58-73 (2009) ] .
  • BCR B cell receptor
  • Btk is activated by its upstream kinases through the phosphorylation of a tyrosine residue (Tyr551) , followed by the autophosphorylation of another tyrosine residue (Tyr223) .
  • the fully activated Btk then phosphorylates its substrates, including PLC- ⁇ 2 in the BCR pathway.
  • Ibrntinib (CRA-032765, PCI-32765, ) , a covalent irreversible inhibitor from Celera/Pharmacyclics/Janssen, became the first clinically approved Btk-targeting drug in November 2013 [Pan, Z. et al. Discovery of selective irreversible inhibitors for Bruton′s tyrosine kinase. ChemMedChem. 2, 58-61 (2007) ] . CC-292 (AVL-292) from Celgene is the second covalent irreversible inhibitor that is currently undergoing clinical trials [Singh, J. et al. inventors; Celgene Avilomics Research, Inc., assignee. 2, 4-Diaminopyrimidines useful as kinase inhibitors. United States patent US 8,609,679. 2013 Dec 17] .
  • Both ibrutinib and CC-292 form a covalent bond with a cysteine residue (Cys481) located at the rim of the ATP-binding pocket in Btk.
  • Other clinical-stage Btk inhibitors include a compound from ONO Pharmaceutical and PRN1008/HM71224 from Hanmi Pharmaceutical [Yamamoto, S. &Yoshizawa T. inventors; Ono Pharmaceutical Co., Ltd., assignee. Purinone derivative. United States patent US 8,940,725. 2015 Jan 27; Hanmi Pharmaceutical Company Limited, Safety, PK/PD, Food Effect Study of Orally Administered HM71224 in Healthy Adult Male Volunteers. Available at: https: //clinicaltrials. gov/ct2/show/NCT01765478 (Accessed: 4th July 2015) ] .
  • GDC-0834 a non-covalent reversible Btk inhibitor from Gilead/Roche, was evaluated in a Phase I clinical trial, but no recent developments have been reported [Liu, L. et al. Significant species difference in amide hydrolysis of GDC-0834, a novel potent and selective Bruton′s tyrosine kinase inhibitor. Drug Metab. Dispos. 39, 1840-1849 (2011) ] .
  • Target engagement refers to the occupancy of intended biological targets by drug molecules [Copeland, R. A., Pompliano, D. L. &Meek, T. D. Drug-target residence time and its implications for lead optimization. Nat. Rev. Drug Discov. 5, 730-739 (2006) ] .
  • This information is crucial for building a correlation between phenotypic observations and inhibitor-biomolecule interactions at the molecular level.
  • Targeted covalent drugs due to their inherent reactive groups, are particularly suitable for developing small molecule affinity probes that may be used to measure the extent of target occupancy [Potashman, M. H. &Duggan, M. E. Covalent modifiers: an orthogonal approach to drug design. J. Med. Chem.
  • PCI-33380 was designed based on the ibrutinib scaffold and has been used in both cellular and in vivo studies that demonstrated the connection between the inhibitor binding event and phenotypic readouts of cellular responses due to the inhibition of Btk functions [Honigberg, L. A. et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc. Natl. Acad. Sci. U. S. A. 107, 13075-13080 (2010) ] .
  • affinity probes normally include three components: a recognition group, a reactive group and a reporting group.
  • the recognition group directs the probe into the binding pocket of the targeted protein and facilitates the formation of a covalent bond between the reactive group and the biomolecule.
  • the reporting group provides a convenient means of identifying probe-bound proteins within complex proteomes.
  • Fig. 4 shows a general scheme of assays to examine the target engagement of drug molecules.
  • R is selected from the group consisting of a bond, carbonylalkyleneamino (e.g., carbonylC 1-6 alkyleneamino) , ( (azacycloalk-2-yl) -alkyl) oxomethane) -l, N-diyl (e.g. ( ( (C 5-7 azacycloalk-2-yl) -C 0-2 alkyl) oxomethane) -1, N-diyl) ; preferably, R is selected from the group consisting of and
  • R 1 is selected from the group consisting of alkyl, arylalkyl, hydroxyalkyl, aminocarbonylalkyl, carboxylalkyl, aminoalkyl and heteroarylalkyl; preferably R 1 is selected from the group consisting of and more preferably, R 1 is selected from the group consisting of and
  • p is 0, 1, 2 or 3, preferably 0;
  • q is 0, 1, 2 or 3, preferably 0;
  • R 11 is a substituent containing an end group selected from OH, COOH, CONH 2 , NH 2 , and a nitrogen containing heterocycle; preferably R 11 is an alkyl group substituted by a substituent selected from the group consisting of OH, COOH, CONH 2 , NH 2 , and a nitrogen containing heterocycle as end group, wherein one or more CH 2 moieties in said alkyl group are optionally replaced with a divalent group selected from the group consisting of-NH-, -CO-, -SO 2 -and -SO-; more preferably, R 11 is selected from the group consisting of and
  • the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) , or a pharmaceutically acceptable salt thereof as the Btk inhibitor for treating B-cell malignancies and autoimmune diseases such as rheumatoid arthritis and lupus.
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) , or a pharmaceutically acceptable salt thereof as the Btk inhibitor, in combination with one or more pharmaceutically acceptable carriers.
  • a Btk affinity probe which comprises a Btk inhibitor moiety, a reporter moiety, and a linker moiety that links the Btk inhibitor moiety to the reporter moiety; wherein the Btk inhibitor moiety is derivable from the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) , or a pharmaceutically acceptable salt thereof.
  • a Btk affinity probe which comprises a Btk inhibitor moiety, a reporter moiety, and a linker moiety that links the Btk inhibitor moiety to the reporter moiety; wherein the Btk inhibitor moiety is derivable from the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) , or a pharmaceutically acceptable salt thereof.
  • Btk affinity probe as represented by the following formula (Ic) :
  • Btk inhibitor moiety is derivable from the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) , or a pharmaceutically acceptable salt thereof;
  • R a is hydrogen or alkyl.
  • a Btk affinity probe wherein the linker moiety covalently links the Btk inhibitor moiety to the reporter moiety.
  • a Btk affinity probe wherein the Btk inhibitor moiety modifies a cysteine residue of a Btk enzyme.
  • a Btk affinity probe wherein the Btk inhibitor moiety covalently modifies the cysteine residue of the Btk enzyme.
  • the cysteine residue is in the ATP binding pocket of the Btk enzyme.
  • a Btk affinity probe wherein the cysteine residue is Cys 481 of the Btk enzyme.
  • a Btk affinity probe wherein the linker moiety is selected from a bond, an optionally substituted alkyl moiety, an optionally substituted heterocycle moiety, an optionally substituted amide moiety, a ketone moiety, an optionally substituted carbamate moiety, an ester moiety, or a combination thereof.
  • the linker moiety is a bond.
  • a Btk affinity probe wherein the reporter moiety is selected from the group consisting of a label, a dye, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, an antibody or antibody fragment, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, a redox-active agent, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chem
  • a Btk affinity probe wherein the reporter moiety is a fluorophore.
  • a Btk affinity probe wherein the fluorophore is a Bodipy fluorophore.
  • a Btk affinity probe wherein the Bodipy fluorophore is a Bodipy FL fluorophore.
  • Btk affinity probe wherein the Btk inhibitor moiety is derived from an irreversible inhibitor of Btk.
  • a Btk affinity probe wherein the irreversible inhibitor of Btk is:
  • Btk affinity probe having the structure:
  • a Btk affinity probe wherein the probe selectively labels a phosphorylated conformation of Btk.
  • a Btk affinity probe wherein the phosphorylated conformation of Btk is either an active or inactive form of Btk.
  • a Btk affinity probe wherein the phosphorylated conformation of Btk is an active form of Btk.
  • the probe is cell permeable.
  • a method for assessing the efficacy of a potential Btk inhibitor in a mammal comprising administering a potential Btk inhibitor to the mammal, administering the Btk affinity probe described herein to the mammal or to cells isolated from the mammal; measuring the activity of the reporter moiety of the Btk affinity probe, and comparing the activity of the reporter moiety to a standard.
  • a method for assessing the pharmacodynamics of a Btk inhibitor in a mammal comprising administering a Btk inhibitor to the mammal, administering the Btk affinity probe presented herein to the mammal or to cells isolated from the mammal, and measuring the activity of the reporter moiety of the Btk affinity probe at different time points following the administration of the inhibitor.
  • a method for in vitro labeling of a Btk enzyme comprising contacting an active Btk enzyme with the Btk affinity probe described herein.
  • the contacting step comprises incubating the active Btk enzyme with the Btk affinity probe presented herein.
  • a method for in vitro labeling of a Btk enzyme comprising contacting cells or tissues expressing the Btk enzyme with an Btk affinity probe described herein.
  • a method for detecting a labeled Btk enzyme comprising separating proteins, the proteins comprising a Btk enzyme labeled by an Btk affinity probe described herein, by electrophoresis and detecting the Btk affinity probe by fluorescence.
  • the present invention includes the following technical solutions:
  • R is selected from the group consisting of a bond, carbonylalkyleneamino, ( ( (azacycloalk-2-yl) -alkyl) oxomethane) -1, N-diyl; preferably R is selected from the group consisting of ( ( (C 5-7 azacycloalk-2-yl) -C 0-2 alkyl) oxomethane) -1, N-diyl) ;
  • R 1 is selected from the group consisting of alkyl, arylalkyl, hydroxyalkyl, aminocarbonylalkyl, carboxylalkyl, aminoalkyl and heteroarylalkyl; preferably R 1 is selected from the group consisting of arylalkyl, hydroxyalkyl, aminocarbonylalkyl, carboxylalkyl, aminoalkyl and heteroarylalkyl; or preferably R 1 is selected from the group consisting of and or preferably R 1 is selected from the group consisting of and
  • p is 0, 1, 2 or 3, preferably 0;
  • q is 0, 1, 2 or 3, preferably 0;
  • R 11 is a substituent containing an end group selected from OH, COOH, CONH 2 , NH 2 , and a nitrogen containing heterocycle; preferably R 11 is an alkyl group substituted by a substituent selected from the group consisting of OH, COOH, CONH 2 , NH 2 , and a nitrogen containing heterocycle as end group, wherein one or more CH 2 moieties in said alkyl group are optionally replaced with a divalent group selected from the group consisting of-NH-, -CO-, -SO 2 -and -SO-; more preferably, R 11 is selected from the group consisting of and
  • Btk inhibitor moiety is derivable from a compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) :
  • R is selected from the group consisting of a bond, carbonylalkyleneamino, ( ( (azacycloalk-2-yl) -alkyl) oxomethane) -1, N-diyl; preferably R is selected from the group consisting of ( ( (C 5-7 azacycloalk-2-yl) -C 0-2 alkyl) oxomethane) -1, N-diyl) ;
  • R 1 is selected from the group consisting of alkyl, arylalkyl, hydroxyalkyl, aminocarbonylalkyl, carboxylalkyl, aminoalkyl and heteroarylalkyl; preferably R 1 is selected from the group consisting of arylalkyl, hydroxyalkyl, aminocarbonylalkyl, carboxylalkyl, aminoalkyl and heteroarylalkyl; or preferably R 1 is selected from the group consisting of and or preferably R 1 is selected from the group consisting of and
  • p is 0, 1, 2 or 3, preferably 0;
  • q is 0, 1, 2 or 3, preferably 0;
  • R 11 is a substituent containing an end group selected from OH, COOH, CONH 2 , NH 2 , and a nitrogen containing heterocycle (for example, the derivation can be made by removing a hydrogen atom from the end group of R 11 ) ; preferably R 11 is an alkyl group substituted by a substituent selected from the group consisting of OH, COOH, CONH 2 , NH 2 , and a nitrogen containing heterocycle as end group, wherein one or more CH 2 moieties in said alkyl group are optionally replaced with a divalent group selected from the group consisting of -NH-, -CO-, -SO 2 -and -SO-; more preferably, R 11 is selected from the group consisting of and
  • R a is hydrogen or alkyl
  • linker moiety is selected from a bond, an optionally substituted alkyl moiety, an optionally substituted heterocycle moiety, an optionally substituted amide moiety, a ketone moiety, an optionally substituted carbamate moiety, an ester moiety, or a combination thereof;
  • the reporter moiety is selected from the group consisting of a label, a dye, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, an antibody or antibody fragment, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, a redox-active agent, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chemiluminescent group, an electron dense group, a
  • Bodipy fluorophore is a Bodipy FL fluorophore.
  • a method for assessing the efficacy of a potential Btk inhibitor in a mammal comprising administering a potential Btk inhibitor to the mammal, administering the Btk affinity probe of any of Technical Solutions 7-12 to the mammal or to cells isolated from the mammal; measuring the activity of the reporter moiety of the Btk affinity probe, and comparing the activity of the reporter moiety to a standard.
  • a method for assessing the pharmacodynamics of a Btk inhibitor in a mammal comprising administering a Btk inhibitor to a plurality of mammals, administering the Btk affinity probe of Technical Solutions 7-12 to the plurality of mammals or to cells isolated from a plurality of mammals, and measuring the activity of the reporter moiety of the Btk affinity probe at different time points following the administration of the inhibitor.
  • a method for in vitro labeling of a Btk enzyme comprising contacting cells or tissues expressing the Btk enzyme with the Btk affinity probe of Technical Solutions 7-12.
  • a method for detecting a labeled Btk enzyme comprising separating proteins, the proteins comprising a Btk enzyme labeled by the Btk affinity probe of Technical Solutions 7-12, by electrophoresis and detecting the Btk affinity probe by fluorescence.
  • alkyl by itself or as part of another molecule means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof.
  • the alkyl chain is fully saturated, mono-or polyunsaturated.
  • the alkyl chain includes di-and multivalent radicals, having the number of carbon atoms designated (i.e. C 0 -C 10 or C 0 - 10 means zero to ten carbons, and C 0 -alkyl means a bond) .
  • examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl) , 2, 4-pentadienyl, 3- (1, 4-pentadienyl) , ethynyl, 1-and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • alkyl, unless otherwise noted, includes those derivatives of alkyl defined in more detail herein, such as “heteroalkyl” , “haloalkyl” and “homoalkyl” .
  • biophysical probe refers to probes which detect or monitor structural changes in molecules.
  • molecules include, but are not limited to, proteins and the “biophysical probe” is used to detect or monitor interaction of proteins with other macromolecules.
  • examples of biophysical probes include, but are not limited to, spin-labels, fluorophores, and photoactivatable groups.
  • carbonyl refers to a group containing a moiety selected from the group consisting of-C (O) -, -S (O) -, -S (O) 2 -, and -C (S) -, including, but not limited to, groups containing a least one ketone group, and/or at least one aldehyde group, and/or at least one ester group, and/or at least one carboxylic acid group, and/or at least one thioester group.
  • Such carbonyl groups include ketones, aldehydes, carboxylic acids, esters, and thioesters. In some embodiments, such groups are a part of linear, branched, or cyclic molecules.
  • chemiluminescent group refers to a group which emits light as a result of a chemical reaction without the addition of heat.
  • luminol 5-amino-2, 3-dihydro-1, 4-phthalazinedione
  • oxidants like hydrogen peroxide (H 2 O 2 ) in the presence of a base and a metal catalyst to produce an excited state product (3-aminophthalate, 3-APA) .
  • chromophore refers to a molecule which absorbs light of visible wavelengths, UV wavelengths or IR wavelengths.
  • Cys 481 refers to the cysteine found in kinases in Fig. 5 at the position corresponding to Cys 481 in Btk (i.e., the “C” highlighted in bold) .
  • the term “detectable label” as used herein refers to a label which is observable using analytical techniques including, but not limited to, fluorescence, chemiluminescence, electron-spin resonance, ultraviolet/visible absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance, and electrochemical methods.
  • die refers to a soluble, coloring substance which contains a chromophore.
  • electrostatic group refers to a group which scatters electrons when irradiated with an electron beam.
  • groups include, but are not limited to, ammonium molybdate, bismuth subnitrate cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate, hexamethylene tetramine, 98.5%, indium trichloride anhydrous, lanthanum nitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate, periodic acid, phosphomolybdic acid, phosphotungstic acid, potassium ferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver proteinate (Ag Assay: 8.0-8.5%) “Strong” , silver tetraphenylporphin (S-TPPS) , sodium chloroaurate, sodium tungstate, thallium nitrate, thiosemicarbazide (S-TPPS) , sodium
  • the term “energy transfer agent” as used herein refers to a molecule which either donates or accepts energy from another molecule.
  • fluorescence resonance energy transfer FRET is a dipole-dipole coupling process by which the excited-state energy of a fluorescence donor molecule is non-radiatively transferred to an unexcited acceptor molecule which then fluorescently emits the donated energy at a longer wavelength.
  • “enhance” or “enhancing” means to increase or prolong either in potency or duration a desired effect.
  • “enhancing” the effect of therapeutic agents refers to the ability to increase or prolong, either in potency or duration, the effect of therapeutic agents on during treatment of a disease, disorder or condition.
  • An “enhancing-effective amount” as used herein refers to an amount adequate to enhance the effect of a therapeutic agent in the treatment of a disease, disorder or condition. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient′s health status and response to the drugs, and the judgment of the treating physician.
  • fluorophore refers to a molecule which upon excitation emits photons and is thereby fluorescent.
  • label refers to a substance which is incorporated into a compound and is readily detected, whereby its physical distribution is detected and/or monitored.
  • linkages as used herein to refer to bonds or a chemical moiety formed from a chemical reaction between the functional group of a linker and another molecule.
  • bonds include, but are not limited to, covalent linkages and non-covalent bonds
  • chemical moieties include, but are not limited to, esters, carbonates, imines, phosphate esters, hydrazones, acetals, orthoesters, peptide linkages, and oligonucleotide linkages.
  • Hydrolytically stable linkages means that the linkages are substantially stable in water and do not react with water at useful pH values, including but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely.
  • Hydrolytically unstable or degradable linkages means that the linkages are degradable in water or in aqueous solutions, including for example, blood.
  • enzymatically unstable or degradable linkages means that the linkage is degraded by one or more enzymes.
  • PEG and related polymers include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule.
  • Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent, wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent.
  • hydrolytically degradable linkages include but are not limited to carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5′hydroxyl group of an oligonucleotide.
  • the phrase “measuring the activity of the reporter moiety” refers to methods for quantifying (in absolute, approximate or relative terms) the reporter moiety in a system under study.
  • such methods include any methods that quantify a reporter moiety that is a dye; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; an antibody or antibody fragment; a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleav
  • molecular incorporating a heavy atom refers to a group which incorporates an ion of atom which is usually heavier than carbon.
  • ions or atoms include, but are not limited to, silicon, tungsten, gold, lead, and uranium.
  • nanoparticle refers to a particle which has a particle size between about 500 nm to about 1 nm.
  • pharmaceutically acceptable refers to a material, including but not limited, to a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic.
  • the material is administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • photoaffinity label refers to a label with a group, which, upon exposure to light, forms a linkage with a molecule for which the label has an affinity.
  • a linkage is covalent or non-covalent.
  • photocaged moiety refers to a group which, upon illumination at certain wavelengths, covalently or non-covalently binds other ions or molecules.
  • photoisomerizable moiety refers to a group wherein upon illumination with light changes from one isomeric form to another.
  • radioactive moiety refers to a group whose nuclei spontaneously give off nuclear radiation, such as alpha, beta, or gamma particles; wherein, alpha particles are helium nuclei, beta particles are electrons, and gamma particles are high energy photons.
  • spin label refers to molecules which contain an atom or a group of atoms exhibiting an unpaired electron spin (i.e. a stable paramagnetic group) that in some embodiments are detected by electron spin resonance spectroscopy and in other embodiments are attached to another molecule.
  • spin-label molecules include, but are not limited to, nitryl radicals and nitroxides, and in some embodiments are single spin-labels or double spin-labels.
  • the phrase ′′therapeutically effective amount′′ of the compound of the invention means a sufficient amount of the compound to treat disorders, at a reasonable benefit/risk ratio applicable to any medical treatment. It is understood, however, that the total daily usage of the compounds and compositions of the invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient depends upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well-known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • pharmaceutically acceptable carrier means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such a propylene glycol; esters such as, but not limited to, ethyl oleate and
  • subject refers to an animal which is the object of treatment, observation or experiment.
  • subject is a mammal including, but not limited to, a human.
  • substituted substituents also referred to as “non-interfering substituents” “refers to groups which are used to replace another group on a molecule. Such groups include, but are not limited to, halo, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, C 1 -C 10 alkoxy, C 5 -C 12 aralkyl, C 3 -C 12 cycloalkyl, C 4 -C 12 cycloalkenyl, phenyl, substituted phenyl, toluolyl, xylenyl, biphenyl, C 2 -C 12 alkoxyalkyl, C 5 -C 12 alkoxyaryl, C 5 -C 12 aryloxyalkyl, C 7 -C 12 oxyaryl, C 1 -C 6 alkylsulfinyl, C 1 -C 10 alkylsulfon
  • each R group in the preceding list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkaryl.
  • substituent groups are specified by their conventional chemical formulas, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left; for example, -CH 2 O-is equivalent to -OCH 2 -.
  • each R group in the preceding list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups.
  • aryl substituted with 1-3 halogens substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups.
  • -NR 2 includes, but is not be limited to, 1-pyrrolidinyl and 4-morpholinyl.
  • Compounds presented herein include isotopically-labeled compounds, which are identical to those recited in the various formulas and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes that are incorporated into the present compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, 36 C1, respectively.
  • isotopically-labeled compounds described herein for example those into which radioactive isotopes such as 3 H and 14 c are incorporated, are useful in drug and/or substrate tissue distribution assays. Further, in other embodiments, substitution with isotopes such as deuterium, i.e., 2 H, affords certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.
  • diastereomeric mixtures are separated into their individual diastereomers on the basis of their physical chemical differences, for example, by chromatography and/or fractional crystallization.
  • enantiomers are separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol) , separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomers, enantiomers, and mixtures thereof are considered as part of the compositions described herein.
  • the compounds described herein are used in the form of pro-drugs. In additional or further embodiments, the compounds described herein are metabolized upon administration to an organism in need to produce a metabolite that is then used to produce a desired effect, including a desired therapeutic effect.
  • the methods and formulations described herein include the use of N-oxides, crystalline forms (also known as polymorphs) , or pharmaceutically acceptable salts.
  • compounds described herein exist as tautomers. All tautomers are included within the scope of the compounds presented herein.
  • the compounds described herein exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the compounds presented herein are also considered to be disclosed herein.
  • salts are prepared by conventional methods.
  • salts are prepared by contacting the acidic and basic entities, in either an aqueous, non-aqueous or partially aqueous medium. The salts are recovered by using at least one of the following techniques: filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.
  • pharmaceutically acceptable salts of the compounds disclosed herein are formed when an acidic proton present in the parent compound either is replaced by a metal ion, by way of example an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base.
  • a metal ion by way of example an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base.
  • the salt forms of the disclosed compounds are prepared using salts of the starting materials or intermediates.
  • the type of pharmaceutical acceptable salts include, but are not limited to: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo- [2.2.2] oc
  • acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.
  • acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.
  • the corresponding counter-ions of the pharmaceutically acceptable salts are analyzed and identified using various methods including, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectroscopy, mass spectrometry, or any combination thereof.
  • a reference to a salt includes the solvent addition forms or crystal forms thereof, particularly solvates or polymorphs.
  • solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and are often formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like.
  • hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol.
  • Polymorphs include the different crystal packing arrangements of the same elemental composition of a compound.
  • polymorphs having different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility.
  • various factors such as the recrystallization solvent, rate of crystallization, and storage temperature cause a single crystal form to dominate.
  • the screening and characterization of the pharmaceutically acceptable salts, polymorphs and/or solvates is accomplished using a variety of techniques including, but not limited to, thermal analysis, x-ray diffraction, spectroscopy, vapor sorption, and microscopy.
  • thermal analysis methods address thermo chemical degradation or thermo physical processes including, but not limited to, polymorphic transitions, and such methods are used to analyze the relationships between polymorphic forms, determine weight loss, to find the glass transition temperature, or for excipient compatibility studies.
  • Such methods include, but are not limited to, Differential scanning calorimetry (DSC) , Modulated Differential Scanning Calorimetry (MDCS) , Thermogravimetric analysis (TGA) , and Thermogravi-metric and Infrared analysis (TG/IR) .
  • DSC Differential scanning calorimetry
  • MDCS Modulated Differential Scanning Calorimetry
  • TGA Thermogravimetric analysis
  • TG/IR Thermogravi-metric and Infrared analysis
  • X-ray diffraction methods include, but are not limited to, single crystal and powder diffractometers and synchrotron sources.
  • the various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid state) .
  • the various microscopy techniques include, but are not limited to, polarized light microscopy, Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX) , Environmental Scanning Electron Microscopy with EDX (in gas or water vapor atmosphere) , IR microscopy, and Raman microscopy.
  • Fig 1 shows structures of representative Btk inhibitors.
  • Fig 2 shows structures of representative fluorescent probes.
  • Fig. 3 shows components of affinity probes
  • Fig. 4 shows general scheme of measuring target engagement by competition assays between inhibitors and affinity probes.
  • Fig. 5 shows sequence alignments of the ATP binding pocket in kinases containing the conserved cysteine (arrow) corresponding to Cys 481 in Btk. The protein accession numbers are also shown.
  • Fig. 6 shows concentration-dependent labeling of recombinant Btk by probe 14.
  • Fig. 7 shows time-dependent labeling of recombinant Btk by probe 14.
  • Fig. 8 shows probe 14 predominantly labeled endogenous Btk in live cells (concentration course) ;
  • Fig. 9 shows the result of time course experiments in cellular labeling.
  • Fig. 10 shows the result of immunoprecipitation of Btk from probe 14-labeled lysates.
  • lane 1 cell lysates
  • lane 2 supernatant after removal of intrinsic IgG
  • lane 3 supernatant after immunoprecipitation
  • lane 4 supernatant of the last wash before elution
  • lane 5 supernatant of the first elution by applying LDS sample buffer onto protein A Sepharose beads.
  • Fig. 11 shows labeling of Btk by probe 14 (0.5 uM) is completely competed off by ibrutinib and compound 2 (1 uM) .
  • Fig. 12 shows the measurement of the extent of Btk occupancy by inhibitors (ibrutinib and compound 2) in live cells. Band densitometry is measured by Gelpro32, and Graphpad Prism is used to determine the IC50 values.
  • Fig. 13 shows the competition experiment in OCI-Ly7 cells (a) and Jurkat cells (b) .
  • 1.5 ⁇ 10 6 cells are pre-incubated with compounds for 1h at 1 ⁇ M before labeling with probe 14 for 2h at 0.5 ⁇ M, then lysed, quantified and analyzed by SDS/PAGE and fluorescent gel scanning (fluorescence, CY2) .
  • Disclosed herein is the synthesis and characterization of Btk inhibitors. Disclosed herein is also the synthesis and characterization of cell permeable probes that label Btk at a unique, non-catalytic cysteine residue in the ATP binding pocket. Other embodiments disclosed herein demonstrate the utility of such probes in assessing pharmacodynamics in mammals treated with small molecule Btk inhibitors.
  • the Btk inhibitor described herein has a general formula (Ia) or (Ib) or (Ib-1) or (Id) , or a pharmaceutically acceptable salt thereof:
  • R is selected from the group consisting of a bond, carbonylalkyleneamino (e.g., carbonylC 1-6 alkyleneamino) , ( (azacycloalk-2-yl) -alkyl) oxomethane) -l, N-diyl (e.g. ( ( (C 5-7 azacycloalk-2-yl) -C 0-2 alkyl) oxomethane) -1, N-diyl) ; preferably, R is selected from the group consisting of and
  • R 1 is selected from the group consisting of alkyl, arylalkyl, hydroxyalkyl, aminocarbonylalkyl, carboxylalkyl, aminoalkyl and heteroarylalkyl; preferably R 1 is selected from the group consisting of and more preferably, R 1 is selected from the group consisting of and
  • p is 0, 1, 2 or 3, preferably 0;
  • q is 0, 1, 2 or 3, preferably 0;
  • R 11 is a substituent containing an end group selected from OH, COOH, CONH 2 , NH 2 , and a nitrogen containing heterocycle; preferably R 11 is an alkyl group substituted by a substituent selected from the group consisting of OH, COOH, CONH 2 , NH 2 , and a nitrogen containing heterocycle as end group, wherein one or more CH 2 moieties in said alkyl group are optionally replaced with a divalent group selected from the group consisting of-NH-, -CO-, -SO 2 -and -SO-; more preferably, R 11 is selected from the group consisting of and
  • Btk inhibitors examples include, but are not limited to:
  • the Btk affinity probe compounds described herein are composed of a moiety comprising an inhibitor of Btk (or a Btk inhibitor moiety) , a linker moiety, and a reporter moiety.
  • the inhibitor of Btk is an irreversible inhibitor.
  • the irreversible inhibitor of Btk binds to a non-catalytic residue in the ATP binding pocket of Btk; in further embodiments, the non-catalytic residue is a cysteine residue.
  • the Btk affinity probe forms a covalent bond with at least one non-catalytic residue of Btk.
  • the Btk affinity probe forms a non-covalent bond with at least one non-catalytic residue of Btk.
  • the Btk affinity probe forms hydrogen bonding within the ATP binding pocket of Btk.
  • the Btk affinity probe has Van der Waals attractions with the Btk enzyme.
  • the Btk affinity probes described herein are activity dependent such that the probe binds only an active Btk enzyme. In further embodiments, the Btk affinity probe binds a Btk enzyme that has been switched on by phosphorylation by upstream kinases. In yet a further embodiment, the Btk affinity probes described herein are activity independent such that the probe binds Btk enzymes that have not been switched on by phosphorylation by upstream kinases. In some embodiments, the Btk affinity probe labels a phosphorylated conformation of a Btk enzyme. In other embodiments, the Btk affinity probe labels a Btk in a non-phosphorylated conformation.
  • the Btk affinity probe is permeable to cells.
  • the linker moiety is selected from a bond, a substituted alkyl moiety, a substituted heterocycle moiety, a substituted amide moiety, a ketone moiety, a substituted carbamate moiety, an ester moiety, or any combination thereof.
  • the reporter moiety is a moiety that is detected using standard or modified laboratory equipment.
  • Btk affinity probe as represented by the following formula (Ic) :
  • Btk inhibitor moiety is derivable from the above-mentioned compound having a general formula (Ia) or (Ib) or (Ib-1) or (Id) ;
  • R a is hydrogen or alkyl.
  • the Btk inhibitor moiety is derived from a Btk inhibitor having a general formula (Ia) or (Ib) or (Ib-1) or (Id) :
  • R is selected from the group consisting of a bond, carbonylalkyleneamino (e.g., carbonylC 1-6 alkyleneamino) , ( (azacycloalk-2-yl) -alkyl) oxomethane) -1, N-diyl (e.g. ( ( (C 5-7 azacycloalk-2-yl) -C 0-2 alkyl) oxomethane) -1, N-diyl) ; preferably, R is selected from the group consisting of and
  • R 1 is selected from the group consisting of alkyl, arylalkyl, hydroxyalkyl, aminocarbonylalkyl, carboxylalkyl, aminoalkyl and heteroarylalkyl; preferably R 1 is selected from the group consisting of and more preferably, R 1 is selected from the group consisting of and
  • p is 0, 1, 2 or 3, preferably 0;
  • q is 0, 1, 2 or 3, preferably 0;
  • R 11 is a substituent containing an end group selected from OH, COOH, CONH 2 , NH 2 , and a nitrogen containing heterocycle; preferably R 11 is an alkyl group substituted by a substituent selected from the group consisting of OH, COOH, CONH 2 , NH 2 , and a nitrogen containing heterocycle as end group, wherein one or more CH 2 moieties in said alkyl group are optionally replaced with a divalent group selected from the group consisting of-NH-, -CO-, -SO 2 -and -SO-; more preferably, R 11 is selected from the group consisting of and
  • Btk inhibitors from which the Btk inhibitor moiety according to the present invention is derived, include, but are not limited to:
  • the Btk inhibitor moiety is selected from the group consisting of
  • the Btk inhibitor moiety is derived from an irreversible inhibitor of Btk.
  • such irreversible inhibitors of Btk should possess at least one of the following characteristics: potency, selectively and cell permeability.
  • such irreversible inhibitors of Btk possess at least two of the aforementioned characteristics, and in further embodiments, at least all of the aforementioned characteristics.
  • the linker moiety is selected from a bond, a polymer, a water soluble polymer, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heterocycloalkylalkenyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocycloalkylalkenylalkyl.
  • the linker moiety is selected from a bond.
  • the linker moiety is an optionally substituted heterocycle.
  • the heterocycle is selected from aziridine, oxirane, episulfide, azetidine, oxetane, pyrroline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, pyrazole, pyrrole, imidazole, triazole, tetrazole, oxazole, isoxazole, oxirene, thiazole, isothiazole, dithiolane, furan, thiophene, piperidine, tetrahydropyran, thiane, pyridine, pyran, thiapyrane, pyridazine, pyrimidine, pyrazine, piperazine, oxazine, thiazine, dithiane, and dioxane.
  • the heterocycle is piperazine.
  • the linker moiety is optionally substituted with halogen, CN, OH, NO 2 , alkyl, S (O) , and S (O) 2 .
  • the water soluble polymer is a PEG group.
  • the linker moiety provides sufficient spatial separation between the reporter moiety and the Btk inhibitor moiety. In further embodiments, the linker moiety is stable. In yet a further embodiment, the linker moiety does not substantially affect the response of the reporter moiety. In other embodiments the linker moiety provides chemical stability to the Btk affinity probe. In further embodiments, the linker moiety provides sufficient solubility to the Btk affinity probe.
  • linkages such as water soluble polymers are coupled at one end to a Btk inhibitor moiety and to a reporter moiety at the other end.
  • the water soluble polymers are coupled via a functional group or substituent of the Btk inhibitor moiety.
  • the water soluble polymers are coupled via a functional group or substituent of the reporter moiety.
  • covalent attachment of hydrophilic polymers to a Btk inhibitor moiety and a reporter moiety represents one approach to increasing water solubility (such as in a physiological environment) , bioavailability, increasing serum half-life, increasing pharmacodynamic parameters, or extending the circulation time of the Btk affinity probe, including proteins, peptides, and particularly hydrophobic molecules.
  • additional important features of such hydrophilic polymers include biocompatibility and lack of toxicity.
  • the polymer is pharmaceutically acceptable for therapeutic use of the end-product preparation.
  • examples of hydrophilic polymers include, but are not limited to: polyalkyl ethers and alkoxy-capped analogs thereof (e.g., polyoxyethylene glycol, polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogs thereof, polyoxyethylene glycol, the latter is also known as polyethylene glycol or PEG) ; polyvinylpyrrolidones; polyvinylalkyl ethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropylmethacrylamide and derivatives thereof) ; polyhydroxyalkyl acrylates; polysialic acids and analogs thereof, hydrophilic peptide sequences; polysaccharides and their derivatives, including dextran and dextran derivatives, e.g., carboxymethyldextran, dex
  • the water soluble polymer is any structural form including but not limited to linear, forked or branched.
  • polymer backbones that are water-soluble, with from 2 to about 300 termini, are particularly useful.
  • multifunctional polymer derivatives include, but are not limited to, linear polymers having two termini, each terminus being bonded to a functional group which is the same or different.
  • the water polymer comprises a poly (ethylene glycol) moiety.
  • the molecular weight of the polymer is of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more.
  • the molecular weight of the polymer is between about 100 Da and about 100,000 Da, including but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da,about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000 Da, about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500 Da, about 400 Da, about 300 Da, about 200 Da, and about 100 Da.
  • the molecular weight of the polymer is between about 100 Da and 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and 40,000 Da. In some embodiments, the poly (ethylene glycol) molecule is a branched polymer.
  • the molecular weight of the branched chain PEG is between about 1,000 Da and about 100,000 Da, including but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da,about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da.
  • the molecular weight of the branched chain PEG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 20,000 Da.
  • the foregoing list for substantially water soluble backbones is by no means exhaustive and is merely illustrative, and in some embodiments, the polymeric materials having the qualities described above suitable for use in methods and compositions described herein.
  • the number of water soluble polymers linked to a Btk inhibitor moiety and a reporter moiety described herein is adjusted to provide an altered (including but not limited to, increased or decreased) pharmacologic, pharmacokinetic or pharmacodynamic characteristic such as in vivo half-life.
  • the half-life of the Btk affinity probe is increased at least about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 percent, about two fold, about five-fold, about 10-fold, about 50-fold, or at least about 100-fold over a Btk affinity probe without a water soluble linker.
  • X is NR a (CO) .
  • X is a bond.
  • X is -O (CO) -.
  • Y is a bond.
  • Y is -NR a (CO) -.
  • R a is hydrogen.
  • R a is alkyl.
  • the reporter moiety is selected from the group consisting of a label, a dye, a photocrosslinker, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, an antibody or antibody fragment, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analog, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, a redox-active agent, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chemiluminescent group, an electron dense group
  • the reporter moiety is a fluorophore.
  • the fluorophore is selected from the group consisting of: BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY TR, Fluorescein, 5 (6) -Carboxyfluorescein, 2, 7-Dichlorofluorescein, N, N-Bis (2, 4, 6-trimethylphenyl) --3, 4: 9, 10-perylenebis (dicarboximide, HPTS, Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green 520, ATTO 465, ATTO 488, ATTO 495, YOYO-1, 5-FAM, BCECF, BCE
  • 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.
  • the fluorophore is BODIPY FL.
  • the fluorophore is not BODIPY 530.
  • the fluorophore has an excitation maxima of between about 500 and about 600 nm.
  • the fluorophore has an excitation maxima of between about 500 and about 550 nm. In another embodiments, the fluorophore has an excitation maxima of between about 550 and about 600 nm. In yet a further embodiment, the fluorophore has an excitation maxima of between about 525 and about 575 nm. In other embodiments, the fluorophore has an emission maxima of between about 510 and about 670 nm. In another embodiment, the fluorophore has an emission maxima of between about 510 and about 600 nm. In a further embodiment, the fluorophore has an emission maxima of between about 600 and about 670 nm. In another embodiment, the fluorophore has an emission maxima of between about 575 and about 625 nm.
  • the linkage formed is a stable linkage.
  • the linker moiety forms a linkage, in some embodiments, a stable linkage, between the Btk inhibitor moiety and the reporter moiety.
  • the linker moiety is stable and provides the means to control and determine the distance between the Btk inhibitor moiety and the report moiety.
  • the linker moiety is selected such that the probe′s solubility is maintained.
  • the number and order of units that comprise the linker moiety is selected such that the length between the first component and the second component, as well as the hydrophobic and hydrophilic characteristics of the linker is controlled.
  • spatial separation means a thermochemically and photochemically non-active distance-making group and in some embodiments is used to join two or more different moieties of the types defined above.
  • spacers are selected on the basis of a variety of characteristics including their hydrophobicity, hydrophilicity, molecular flexibility and length.
  • the spacer thus, in some embodiments, comprises a chain of carbon atoms optionally interrupted or terminated with one or more heteroatoms, such as oxygen atoms, nitrogen atoms, and/or sulphur atoms.
  • the spacer comprises one or more amide, ester, amino, ether, and/or thioether functionalities, and optionally aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly-. ⁇ -alanine, polyglycine, polylysine, and peptides in general, oligosaccharides, oligo/polyphosphates.
  • the spacer consists of combined units thereof.
  • the length of the spacer varies, taking into consideration the desired or necessary positioning and spatial orientation of the active/functional part of the Btk affinity probe.
  • the reporter moiety is Bodipy.
  • reporter moiety means a group which is detectable either by itself or as a part of a detection series.
  • Compound (14) retains the solubility and membrane permeability of Compound (11) , allowing detection and quantitation of labeled Btk by SDS-PAGE and laser densitometry.
  • the labeled Btk affinity probes described herein are purified by one or more procedures including, but are not limited to, affinity chromatography; anion-or cation-exchange chromatography (using, including but not limited to, DEAE SEPHAROSE) ; chromatography on silica; reverse phase HPLC; gel filtration (using, including but not limited to, SEPHADEX G-75) ; hydrophobic interaction chromatography; size-exclusion chromatography, metal-chelate chromatography; ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfate precipitation; chromatofocusing; displacement chromatography; electrophoretic procedures (including but not limited to preparative isoelectric focusing) , differential solubility (including but not limited to ammonium sulfate precipitation) , or extraction.
  • affinity chromatography anion-or cation-exchange chromatography (using, including but not limited to, DEAE SEPHAROSE) ; chromatography on silica; reverse phase HPLC;
  • apparent molecular weight is estimated by GPC by comparison to globular protein standards (PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH (Harris &Angal, Eds. ) IRL Press 1989, 293-306) .
  • the synthetic procedures disclosed below includes various purifications, such as column chromatography, flash chromatography, thin-layer chromatography (TLC) , recrystallization, distillation, high-pressure liquid chromatography (HPLC) and the like.
  • various techniques for the identification and quantification of chemical reaction products such as proton and carbon-13 nuclear magnetic resonance ( 1 H and 13 C NMR) , infrared and ultraviolet spectroscopy (IR and UV) , X-ray crystallography, elemental analysis (EA) , HPLC and mass spectroscopy (MS) are used as well.
  • Example 4 A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with 3- (tert-butoxycarbonylamino) propanoic acid in step 1, Example 4 to produce the title compound as white solids.
  • Example 4 A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -1- (tert-butoxycarbonyl) pyrrolidine-2-carboxylic acid in step 1, Example 4 to produce the title compound as white solids.
  • Example 4 A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -2- (tert-butoxycarbonylamino) propanoic acid in step 1, Example 4 to produce the title compound as white solids.
  • Example 4 A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -2- (tert-butoxycarbonylamino) -3-phenylpropanoic acid in step 1, Example 4 to produce the title compound as white solids.
  • Example 4 A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -2- (tert-butoxycarbonylamino) -3-hydroxypropanoic acid in step 1, Example 4 to produce the title compound as light yellow solids.
  • Example 4 A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -4-amino-2- (tert-butoxycarbonylamino) -4-oxobutanoic acid in step 1, Example 4 to produce the title compound as light yellow solids.
  • Example 4 A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -2- (tert-butoxycarbonylamino) pentanedioic acid in step 1, Example 4 to produce the title compound as light yellow solids.
  • Example 4 A procedure similar to Example 4 was repeated except for replacing N- (tert-Butoxycarbonyl) -L-leucine with (S) -2- (tert-butoxycarbonylamino) -3- (1H-imidazol-5-yl) propanoic acid in step 1, Example 4 to produce the title compound as light yellow solids.
  • Kinases were purchased from Carna Biosciences. Kinase enzymology assays were performed according to the protocols specified for the KinEase TM assays sold by Cisbio Bioassays.
  • the kinase assays were performed at room temperature.
  • the compounds with serial dilution in DMSO were added into reaction buffer with 0.5nM Btk, incubating with different periods of time (0min, 4min, 8min, 12min, 16min, and 20min) .
  • Enzyme reaction was started by adding ATP and substrate to the reaction mixture.
  • the enzyme activity was measured with KinEase TM assays.
  • the data analysis was guided by the book, Enzyme Kinetics by Hans Bisswanger [Bisswanger, H. Enzyme Kinetics -principles and methods, 103-106 (Weinheim, 2002) ] .
  • LY7 cells were treated with probe 14 at 0.5 ⁇ M for 2h, lysed in binding buffer (20 mM Na 3 PO 4 , pH 7.5, 150 mM NaCl) containing phosphatase inhibitors and protease inhibitors. Obtained lysates was preincubated with Protein A Sepharose beads (GE healthcare, 17-5138-01) to remove intrinsic cellular IgG proteins. Meanwhile, anti-Btk (CST, 8574S) from rabbit was preincubated with Protein A Sepharose beads for 2 hours at 4°C. Then, the pre-treated lysates were added to the pre-treated immobilized Protein A Sepharose, incubated for 2 hours at 4°C.
  • binding buffer 20 mM Na 3 PO 4 , pH 7.5, 150 mM NaCl
  • the immune complexes were washed with binding buffer for four times and eluted with LDS sample buffer (50 mM Tris-HCl, 2%SDS, 0.1%bromophenol blue, 10%glycerol, 1%DTT) and analyzed by Western blot, as described above.
  • LDS sample buffer 50 mM Tris-HCl, 2%SDS, 0.1%bromophenol blue, 10%glycerol, 1%DTT
  • LY7 cells were preincubated with the compounds (1 ⁇ M) for 1 h before labeling with probe 14 under the proper time and concentration conditions. Then, the cells were lysed and analyzed as described above.
  • LY7 cells were preincubated with different concentrations of compounds for 1 h before labeling with probe 14. Then, the samples were lysed and analyzed as described above. Gelpro32 software was used to analyze the Btk band density to obtain the half-maximum active site occupancy values.
  • Compound 14 is a selective affinity probe for Btk
  • Btk was indeed the dominant band labeled by probe 14 in OCI-Ly7 cells (Fig. 13a) .
  • Fig. 13b As expected, no significant labeling was detected in Jurkat cells, a T-cell line that does not express Btk (Fig. 13b) .
  • probe 14 After the labeling conditions were optimized, it was examined whether probe 14 could be used to access the target engagement of inhibitors towards Btk.
  • Two types of structurally different Btk inhibitors were examined: the clinically approved Btk drug ibrutinib and compound 2, which contains the same scaffold as probe 14. Cells were first incubated with the inhibitors for 1 hour, followed by 2 hours incubation with 0.5 uM of probe 14. As shown in Fig. 11, both compounds at 1 uM effectively blocked the labeling of Btk by probe 14.
  • OCI-Ly7 cells were incubated with increasing concentrations of the compounds for 1 hour prior to labeling with probe 14 for 2 hours. After cell lysis, the protein contents were directly loaded onto gels. After electrophoresis, the bands’ fluorescent densities were measured. As presented in Fig. 12, the IC 50 values for Btk occupancy by ibrutinib and compound 2 were 2 nM and 8 nM, respectively.

Abstract

La présente invention concerne des inhibiteurs de la tyrosine kinase de Bruton à base de 2,5-diaminopyrimidine. La présente invention concerne également des sondes d'affinité à base de 2,5-diaminopyrimidine pour la tyrosine kinase de Bruton, et les utilisations de ces sondes pour mesurer l'activité de la Btk, pour évaluer l'activité de modulateurs de Btk, et pour évaluer les propriétés pharmacocinétiques et pharmacodynamiques de ces modulateurs.
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