EP3980126A1 - Benzo[h][1,6]naphthyridin-2(1h)-one als bmx-inhibitoren zur verwendung gegen krebs - Google Patents

Benzo[h][1,6]naphthyridin-2(1h)-one als bmx-inhibitoren zur verwendung gegen krebs

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Publication number
EP3980126A1
EP3980126A1 EP20732523.4A EP20732523A EP3980126A1 EP 3980126 A1 EP3980126 A1 EP 3980126A1 EP 20732523 A EP20732523 A EP 20732523A EP 3980126 A1 EP3980126 A1 EP 3980126A1
Authority
EP
European Patent Office
Prior art keywords
compound
group
formula
covalent bond
bmx
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP20732523.4A
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English (en)
French (fr)
Inventor
João SEIXAS
Gonçalo BERNARDES
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Instituto de Medicina Molecular Joao Lobo Antunes
Original Assignee
Instituto de Medicina Molecular Joao Lobo Antunes
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Application filed by Instituto de Medicina Molecular Joao Lobo Antunes filed Critical Instituto de Medicina Molecular Joao Lobo Antunes
Publication of EP3980126A1 publication Critical patent/EP3980126A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the present invention provides compounds having a tricyclic core of a quinoline fused to a pyridinone, and pharmaceutical compositions comprising such compounds.
  • the compounds and compositions find use in methods of treatment, such as methods of treating cancer.
  • complexes comprising a compound of the invention covalently bound to a polypeptide, such as covalently bound to BMX, and polymorphs of these complexes.
  • ETK epithelial and endothelial tyrosine kinase
  • BMX bone marrow tyrosine kinase in chromosome X
  • BMX is a major member of the TEC family of non-receptor tyrosine kinases, together with ITK, TEC, BTK and TXK (reviewed by Smith et al. and Horwood et al.).
  • TEC kinases are activated by many cell surface receptor-associated signalling complexes and are recruited to the plasma membrane or specific micro-environments by a variety of lipids and proteins. Through this mechanism, they are involved in signal transduction in response to a myriad of extracellular stimuli, including those mediated by growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen-receptors, integrins and death receptors.
  • TEC kinases regulate many of the major signalling pathways, such as those for PI3K, PKC, PLCy, AKT, STAT3 and p-activated kinase 1 (PAK1) (see Jarboe et al. and Giu et al.) while being responsible for a variety of cell processes, including regulation of gene expression, calcium mobilization, actin reorganization/motility and survival/apoptosis (Smith et al. and Horwood et al.).
  • PAK1 p-activated kinase 1
  • BMX is widely expressed in granulocytes, monocytes, cells of epithelial and endothelial lineages, as well as brain, prostate, lung and heart and it is specifically involved in tumorigenicity, adhesion, motility, angiogenesis, proliferation and differentiation (see
  • BMX is also required for stem cell maintenance and survival (see Kaukonen et al.) and its up-regulation gives a survival benefit to primary tumours and the cancer stem cells which are highly resistant to apoptosis and many chemotherapeutic agents.
  • Homozygous BMX knockout mice have a normal life span without any obvious altered phenotype, suggesting that therapies based on BMX inhibition might have few side effects (Rajantie et al.).
  • EGFR Endothelial Growth Factor Receptor
  • BMX-IN-1 is one of the most potent BMX inhibitors (IC 50 : 8.0 nM) reported in the literature and also binds to BTK with very high affinity (IC 50 : 10.4 nM) (Liu et al. ACS Chem. Biol.
  • WO 2014/063054 describes compounds for use as inhibitors of Bone Marrow Tyrosine Kinase on Chromosome on X (BMX).
  • BMX Bone Marrow Tyrosine Kinase on Chromosome on X
  • This includes compounds having a tricyclic core of a quinoline fused to a pyridinone at positions 3 and 4 of the quinoline.
  • the core is substituted at the pyridinone ring nitrogen atom, and is further substituted at the 6-position of the quinoline ring.
  • the 6-substituent contains a phenyl group, which may be connected directly to the 6-position of the quinoline ring, or may be connected via a Ci-e hydrocarbon linker, such as an ethylene linker (-CHCH-).
  • Certain compounds exemplified in WO 2014/063054 are said to bind other kinases, such as BTK, mTOR, BLK, TEC, TAK1 , CLK1/2 and JAK3.
  • WO 2014/063054 notes that the disclosed compounds have antiproliferative activity, and are therefore suitable for use in treating cancerous cells, such as WM and lymphoma cell lines.
  • Liu et al. discloses a compound for use as an inhibitor of BMX.
  • the compound has a tricyclic core of a quinoline fused to a pyridinone at positions 3 and 4 of the quinoline.
  • the core is substituted at the pyridinone ring nitrogen atom, and is further substituted at the 6-position of the quinoline ring.
  • the 6-substituent is a phenyl group which is substituted with sulfonamide.
  • the disclosed compound is shown to have antiproliferative activity against panel of prostate cancer cell lines
  • Wu et al. discloses a compound for use as an inhibitor of Bruton’s tyrosine kinase (BTK).
  • the compound has a tricyclic core of a quinoline fused to a pyridinone, similar to the tricyclic cores disclosed in WO 2014/063054 and Liu et al.
  • the 6-substituent to the quinoline ring is a pyrazolyl group.
  • the disclosed compound is shown to suppress the inflammatory response in a rheumatoid arthritis model.
  • Wang et al. discloses compounds for use as inhibitors of BTK.
  • the compound has a tricyclic core of a quinoline fused to a pyridinone, similar to the tricyclic cores disclosed in
  • the 6-substituent to the quinoline ring is an aromatic group, such as phenyl, with the exception of one example compound where a phenyl group is attached to the 6-position via an ethylene linker.
  • the present invention provides compounds having a quinoline ring fused to a pyridinone, and more specifically a 2-pyridinone, at positions 3 and 4 of the quinoline.
  • the core is substituted at the pyridinone ring nitrogen atom with a cyclic group, and is further substituted at the 7-position of the quinoline ring.
  • the 6-substituent does not contain an aromatic group connected to the quinoline ring, or the 6-substituent does not contain an aromatic group connected to the quinoline ring via a Ci- 6 hydrocarbon linker.
  • the compounds of the invention may have improved binding to BMX and other kinases compared with compounds known in the art.
  • the compounds of the invention may also have unexpected binding to BMX and other kinases, taking into account the teaching in the art, particularly with regards to the binding modes predicted by the prior art.
  • the compounds of the invention may have an altered, such as improved, selectively to kinases, such as BMX, or an optimized physicochemical profile.
  • the compounds of the invention are suitable for forming complexes with kinases such as BMX, and such complexes are crystallisable.
  • the crystal structures provide an insight into the mode of binding and may be used for the future development of inhibitors with improved efficacy and selectivity.
  • -A- is an optionally substituted cyclic group selected from arylene, cycloalkylene and heterocycylene, which cyclic group may be fused to a further ring;
  • -L- is a covalent bond or Ci-e alkylene
  • -D is an acceptor group, such as a Michael acceptor group
  • -R 7 is -L 7A -L 7B -R 7A , where
  • -L 7A - is a covalent bond, or is selected from *-0-, *-S-, *-NH-, *-N(R N )-, *-C(0)-, *-C(0)NH-, *-C(0)N(R N )-, *-NHC(0)-, *-N(R N )C(0)-, *-S(0) 2 NH-, *-S(0) 2 N(R N )-, *-NHS(0) 2 - and *-N(R N )S(0) 2 -, where -R N is Ci-e alkyl and the asterisk indicates the point of attachment to the quinoline;
  • -L 7B - is selected from a covalent bond or selected from Ci-e alkylene
  • -R 7A is selected from optionally substituted cycloalkyl, heterocyclyl, and aryl, and when -L 7B - is a covalent bond, -R 7A is further selected from optionally substituted alkyl, alkenyl, alkynyl and heteroalkyl.
  • -A- is an optionally substituted cyclic group selected from arylene, cycloalkylene and heterocyclylene, which cyclic group may be fused to a further ring;
  • -L- is a covalent bond or Ci-e alkylene
  • -D is an acceptor group, such as a Michael acceptor group
  • -R 6 is -
  • -L 6B - is a covalent bond or is selected from Ci-e alkylene, C 2-6 alkenylene,
  • -R 6A is selected from optionally substituted cycloalkyl and heterocyclyl, and when -L 6B - is a covalent bond, -R 6A is further selected from optionally substituted alkyl, alkenyl, alkynyl and heteroalkyl.
  • the compounds of formula (II) are not either of the following compounds:
  • a pharmaceutical composition comprising a compound of formula (I) according to the first aspect of the invention, or a compound of formula (II) according to the second aspect of the invention, together with a pharmaceutically acceptable carrier.
  • the present invention provides the use of a compound of formula (I) according to the first aspect of the invention, the use of a compound of formula (II) according to the second aspect of the invention, or the use of pharmaceutical composition according to the third aspect of the invention, in a method of treatment of the human or animal body.
  • the present invention provides the use of a compound of formula (I) according to the first aspect of the invention, the use of a compound of formula (II) according to the second aspect of the invention, or the use of pharmaceutical composition according to the third aspect of the invention, in a method of treating a proliferative disease, such as cancer.
  • the present invention provides the use of a compound of formula (I) according to the first aspect of the invention, the use of a compound of formula (II) according to the second aspect of the invention, or the use of pharmaceutical composition according to the third aspect of the invention, in a method of treating an autoimmune disease, such as rheumatoid arthritis or lupus.
  • the present invention also provides methods of treatment of a human or animal body. Such methods make use of the compounds and the compositions of the invention as described above, which may be administered in a therapeutically effective amount to a subject.
  • the invention also provides a method of treating a cell, the method comprising the step of contacting a cell with a compound of formula (I) according to the first aspect of the invention or a compound of formula (II) according to the second aspect of the invention.
  • the cell may be a proliferative cell, such a cancer cell.
  • the method may be performed in vitro or in vivo.
  • the present invention provides a complex of a polypeptide covalently bound to a compound of formula (I) according to the first aspect of the invention, or a compound of formula (II) according to the second aspect of the invention.
  • a method of forming a complex comprising the step of reacting a compound of formula (I) according to the first aspect of the invention, or a compound of formula (II) according to the second aspect of the invention, with a polypeptide.
  • polypeptide may be a protein, such as a kinase, such as BMX or BTK.
  • Figure 1 shows the change in BMX IC50 values for compounds 9-29 compared with
  • BMX-IN-1 Human recombinant BMX was incubated with the compounds and
  • phosphorylation of a biotinylated peptide measured by HTRF Values are expressed in potency gain or loss (fold) against control BMX-IN-1 used in each set of experiments. The full library was tested in 4 different experiments, where BMX-IN-1 was always used as control. Compound 10 had a potency loss of 58-fold, while compounds 11-13 had a potency loss of more than 275-fold.
  • Figure 2 shows the mass spectrometric analysis of BMX together with a drug conjugated BMX.
  • A Native MS analysis of hBMX. The measured molecular weight is indicated.
  • B Denaturing MS analysis of drug conjugated hBMX. The measured molecular weight is indicated.
  • C Tandem MS analysis of the drug conjugated tryptic peptide of hBMX labeled on the sequence (top) and MS/MS mass spectrum (bottom). The red asterisk indicates the drug conjugated Cys.
  • Figure 3 shows the co-crystal structure exhibiting the binding mode of 24 to hBMX kinase catalytic domain. A) Covalent bond between the acrylamide of 24 and Cys496. B) Non bonding interactions between 24 and Lys445 and Ile492. C) DFG-motif adopting the out-like conformation (D) Positioning of the sulfonamide aromatic ring pointing out of the ATP pocket.
  • Figure 4 show the flow cytometry results for the use of BMX-IN-1 and compounds 24-27 in an apoptosis study in LNCaP prostate cancer cells.
  • Figure 5 shows the anti-proliferative activity in LNCaP cells of compounds 24-26 in combination with AKT1/2 (AKT inhibitor), Flutamide (androgen receptor antagonist) and LY293002 (PI3K inhibitor).
  • Figure 6 shows the induced targeted cell cytotoxicity on the B-cancer cell in primary DLBCL samples.
  • RCF Relative cell fraction
  • the compounds of the present invention are suitable for forming covalent bonds to kinases such as BMX.
  • the compounds of the invention possess a tricyclic core of a quinoline fused to a pyridinone, and more specifically a 2-pyridinone, at positions 3 and 4 of the quinoline.
  • the core is substituted at the pyridinone ring nitrogen atom, and is further substituted at the 6- or 7-positions of the quinoline ring.
  • the compounds previously described in the art possess a tricyclic core of a quinoline fused to a pyridinone at positions 3 and 4 of the quinoline, and the core is further substituted at the 6-position of the quinoline ring.
  • the substituent that is present at the 6-position of the known compounds is an aromatic group connected to the quinoline ring, either directly or via a Ci- 6 hydrocarbon linker.
  • the compounds of formula (I) differ from those compounds known in the prior art in that they are substituted at the 7-position of the quinoline ring rather than the 6-position.
  • the compounds of formula (II) differ from those compounds known in the prior art in that they are substituted at the 6-position with a substituent that does not contain an aromatic group connected to the quinoline ring, either directly or via a Ci-e hydrocarbon linker.
  • the invention provides a compound of formula (I):
  • -A- is an optionally substituted cyclic group selected from arylene, cycloalkylene and heterocycylene, which cyclic group may be fused to a further ring;
  • -L- is a covalent bond or Ci-e alkylene
  • -D is an acceptor group, such as a Michael acceptor group
  • -R 7 is -L 7A -L 7B -R 7A ,
  • -L 7A - is a covalent bond, or is selected from *-0-, *-S-, *-NH-, *-N(R N )-, * -C(0)-, * -C(0)NH-, * -C(0)N(R N )-, * -NHC(0)-, * -N(R N )C(0)-, * -S(0) 2 NH-, * -S(0) 2 N(R N )-, * -NHS(0) 2 - and * -N(R N )S(0) 2 -, where -R N is Ci-e alkyl and the asterisk indicates the point of attachment to the quinoline;
  • -L 7B - is a covalent bond or selected from Ci-e alkylene, C2-6 alkenylene,
  • -R 7A is selected from optionally substituted cycloalkyl, heterocyclyl, and aryl, and when -L 7B - is a covalent bond, -R 7A is further selected from optionally substituted alkyl, alkenyl, alkynyl and heteroalkyl.
  • the substituent groups and the optional substituents groups for -R 7A are described in further detail below.
  • the invention also provides a compound of formula (I):
  • -A- is an optionally substituted cyclic group selected from arylene, cycloalkylene and heterocyclylene, which cyclic group may be fused to a further ring;
  • -L- is a covalent bond or Ci-e alkylene
  • -D is an acceptor group, such as a Michael acceptor group
  • -R 6 is -L 6A -L 6B -R 6A ,
  • -L 6A - is a covalent bond or is selected from *-0-, *-S-, *-NH-, *-N(R N )-, *-C(0)-, * -C(0)NH-, * -C(0)N(R N )-, * -NHC(0)-, * -N(R N )C(0)-, * -S(0) 2 NH-, * -S(0) 2 N(R N )-, * -NHS(0) 2 - and *-N(R N )S(0) 2 -, where -R N is Ci-e alkyl and the asterisk indicates the point of attachment to the quinoline;
  • -L 6B - is a covalent bond or is selected from Ci-e alkylene, C 2-6 alkenylene,
  • -R 6A is selected from optionally substituted cycloalkyl and heterocyclyl, and when -L 6B - is a covalent bond, -R 6A is further selected from optionally substituted alkyl, alkenyl, alkynyl and heteroalkyl.
  • the compound of formula (II) may not be:
  • the compound of formula (II) may not be:
  • the group -A- is a cyclic group which is a substituent at the nitrogen ring atom of the pyridinone ring within the tricyclic core.
  • phenylene as the cyclic group -A-. It is known from the art that other cyclic groups may be used at this position.
  • WO 2014/063054 describes compounds having a range of cyclic and bicyclic groups, including phenylene, pyridinene, tetrahydroquinolinylene and tetrahydroisoquinolinylene amongst others (these are the groups -C- and -F- in this prior art disclosure).
  • the compounds of the present case are not limited to the use of a phenylene group at -A-.
  • the cyclic group is substituted with the group -L-D, and it is optionally further substituted, for example with one or more groups -R A .
  • Each cyclic group may be monocyclic or may be a series of fused rings, such as a bicyclic ring.
  • the group -A- may be a cyclic group selected from arylene, cycloalkylene and
  • heterocyclylene which cyclic group may be fused to a further ring.
  • Each cyclic group is optionally substituted with one or more substituents -R A .
  • the cyclic group is substituted with one further substituent, -R A .
  • the group -A- is preferably a cyclic group having 6, 9 or 10 ring atoms only.
  • Each of the ring atoms may be a carbon ring atom, and optionally one of the ring atoms may be a nitrogen ring atom.
  • -A- comprises two or more fused rings
  • the ring attached to the nitrogen ring atom of the pyridone group is a 6-membered ring.
  • the ring that is fused to the 6-membered ring is preferably a 5- or a 6-membered ring.
  • -A- is arylene
  • this may be carboarylene or heteroarylene.
  • the arylene may be monocyclic, or may comprise a plurality of fused rings. Where a plurality of rings is present, the ring connected to the pyridone group is aromatic. The other rings are optionally aromatic. The other rings may be fully unsaturated or partially unsaturated. The other rings may be independently selected from aromatic, cycloalkyl and heterocyclyl rings.
  • a carboarylene group may be selected from phenylene (Ob carboarylene), naphthylene and tetralinylene (Cio arylene).
  • a heteroarylene group may be C5-10 heteroarylene, such as C5-6 heteroarylene.
  • the heteroarylene may be selected from pyridinylene (Ob); indolylene, isoindolylene, benzoimidazolylene, indolinylene and isoindolinylene (Cg); and tetrahydroquinolinylene and tetrahydroisoquinolinylene (C10).
  • -A- is cycloalkylene this may be C3-10 cycloalkylene, such as C5-10 alkylene.
  • the cycloalkylene may be monocyclic, or may comprise a plurality of fused rings.
  • cycloalkylene group may be partially unsaturated (but not aromatic).
  • the ring connected to the pyridone group is non aromatic, and is preferably a fully saturated ring.
  • the other rings are optionally aromatic.
  • the other rings may be fully unsaturated, partially unsaturated or saturated.
  • the other rings may be independently selected from aromatic, cycloalkyl and heterocyclyl rings.
  • a cycloalkylene group may be selected from cyclopentylene (C 5 ), cyclohexylene (Ob);
  • tetralinylene and decalinylene such as cyclohexylene.
  • -A- is heterocyclylene this may be C3-10 heterocyclylene, such as
  • a heterocyclylene has one or two ring heteroatoms, with each ring heteroatom selected from O, S and N(H). The ring heteroatom is not connected to the nitrogen ring atom of the pyridone.
  • a heterocyclylene group may be partially unsaturated (but not aromatic).
  • the heterocyclylene may be monocyclic, or may comprise a plurality of fused rings. Where a plurality of rings is present, the ring connected to the pyridone group is non-aromatic, and is preferably a fully saturated ring.
  • the other rings are optionally aromatic.
  • the other rings may be fully unsaturated, partially unsaturated or saturated.
  • the other rings may be independently selected from aromatic, cycloalkyl and heterocyclyl rings.
  • a heterocyclylene may be selected from pyrrolidinylene, tetrahydorfuranylene,
  • tetrahydropyranylene dioxanylene, thianylene, dithianylene, morpholinylene and thiomorpholinylene (Ob); indolinylene, decahydroisoquinolinylene, decahydroquinolinylene and tetrahydroquinoline, and tetrahydroisoquinoline (C10).
  • the group -L-D is preferably provided at the 3-position (where the 1 -postion is the point of attachment to the nitrogen ring atom of the pyridone group).
  • the group -L-D is preferably provided at the 3-position and any further substituents -R A may be provided at one or more of the 2-, 4-, 5- and 6-positions (again, where the 1 -postion is the point of attachment to the nitrogen ring atom of the pyridone group).
  • the cyclic group is not substituted at the 2- or 6-positions.
  • the substituent is provided at the 4-position. It is most preferred that a group -R A is provided at the 4-position and the group -L-D is provided at the 3-position.
  • the group -A- is an optionally substituted group selected from phenylene, pyridinylene, indolylene, isoindolylene, benzoimidazolylene, indolinylene, isoindolinylene, tetrahydroquinolinylene and tetrahydroisoquinolinylene.
  • the group -A- is an optionally substituted group selected from phenylene, pyridinylene, indolylene, 1 ,2,3,4-tetrahydroquinolinylene and indolinylene.
  • the cyclic group is connected to both the pyridone of the tricyclic core and -L-.
  • the cyclic group may be optionally further substituted, such as substituted with one, two, three or four further substituents -R A .
  • -A- is phenylene, this may be phenyl-1 , 3-ene.
  • the 1-position is the carbon ring atom attached to the pyridone nitrogen.
  • phenylene is substituted with -R A these may be provided at one or more of the 2-, 4-, 5- and 6-positions, and preferably at one or more of the 4- and 5-positions, as noted above.
  • the phenylene is monosubstituted, and is substituted at the 4-position.
  • -A- is pyridinylene
  • this may be a pyridinylene selected from the group consisting of pyridinyl-2, 3-ene, pyridinyl-2,4-ene, pyridinyl-2,5-ene, pyridinyl-2,6-ene, pyridinyl-3,4-ene, and pyridinyl-3,5-ene.
  • the 1-position is the nitrogen ring atom.
  • the pyridinylene is unsubstituted or monosubstituted with -R A .
  • the pyridinylene may be substituted at a carbon ring atom that is at the 2-, 3-, 4-, 5- or 6-position, where that position is available for substitution.
  • -A- is indolylene this may be indolyl-1 ,6-ene, where the 1 -position is nitrogen ring atom.
  • the indolylene may be connected to -L- via the nitrogen ring atom.
  • the indolylene may be connected to pyridine via the 6-position.
  • the indolylene is unsubstituted or monosubstituted with -R A .
  • the indolylene is preferably substituted on the benzene ring with -R A .
  • -A- is isoindolylene this may be isoindolyl-2,5-ene, where the 2-position is nitrogen ring atom.
  • the isoindolylene may be connected to -L- via the nitrogen ring atom.
  • the isoindolylene may be connected to pyridine via the 5-position.
  • the isoindolylene is unsubstituted or monosubstituted with -R A .
  • the isoindolylene is preferably substituted on the benzene ring with -R A .
  • -A- is benzoimidazolylene this may be benzoimidazolyl-1 ,6-ene, where the 1 -position is nitrogen ring atom.
  • the benzoimidazolylene may be connected to -L- via the nitrogen ring atom.
  • the benzoimidazolylene may be connected to pyridine via the 6-position.
  • the benzoimidazolylene is unsubstituted or monosubstituted with -R A .
  • benzoimidazolylene is preferably substituted on the benzene ring with -R A .
  • -A- is indolinylene this may be indolinyl-1 ,6-ene, where the 1-position is nitrogen ring atom.
  • the indolinylene may be connected to -L- via the nitrogen ring atom.
  • the indolinylene may be connected to pyridine via the 6-position.
  • the indolinylene is unsubstituted or monosubstituted with -R A .
  • the indolinylene is preferably substituted on the benzene ring with -R A .
  • -A- is isoindolinylene this may be isoindolinyl-2,5-ene, where the 2-position is nitrogen ring atom.
  • the isoindolinylene may be connected to -L- via the nitrogen ring atom.
  • the isoindolinylene may be connected to pyridine via the 5-position.
  • the isoindolinylene is unsubstituted or monosubstituted with -R A .
  • the isoindolinylene is preferably substituted on the benzene ring with -R A .
  • -A- is tetrahydroquinolinylene (or 1 ,2,3,4-tetrahydroquinolinylene) this may be tetrahydroquinolinyl-1 ,7-ene, where the 1-position is nitrogen ring atom.
  • tetrahydroquinolinylene may be connected to -L- via the nitrogen ring atom.
  • the tetrahydroquinolinylene may be connected to pyridine via the 7-position.
  • tetrahydroquinolinylene is unsubstituted or monosubstituted with -R A .
  • tetrahydroquinolinylene is preferably substituted on the benzene ring with -R A .
  • -A- is tetrahydroisoquinolinylene (or 1 ,2, 3, 4- tetrahydroisoquinolinylene) this may be tetrahydroisoquinolinyl-2,6-ene, where the 1-position is nitrogen ring atom.
  • tetrahydroisoquinolinylene may be connected to -L- via the nitrogen ring atom.
  • the tetrahydroisoquinolinylene may be connected to pyridine via the 6-position.
  • tetrahydroisoquinolinylene is unsubstituted or monosubstituted with -R A .
  • tetrahydroisoquinolinylene is preferably substituted on the benzene ring with -R A .
  • the group -A- is optionally substituted phenylene, such as phenylene substituted with one further substituent -R A .
  • the phenylene is phenyl-1 , 3-ene, optionally substituted at the 4-position.
  • the 1 -position is the carbon ring atom attached to the pyridone nitrogen.
  • the group -A- may be optionally substituted indolinylene, such as indolinyl-1 ,6-ene, where the 1-position is nitrogen ring atom.
  • the indolinylene is preferably unsubstituted.
  • the group -R A is a substituent to the cyclic group -A-.
  • the cyclic group -A- may have one or more substituents, each of which is -R A .
  • the cyclic group -A- is not substituted with -R A , it is monosubstituted with -R A or it is disubstituted with -R A .
  • the cyclic group is not substituted with -R A , or it is monosubstituted with -R A .
  • the group -A- is not further substituted or is further substituted with methyl. It is known from the art that other substituent groups may be provided to the group -A-, whilst maintaining biological activity. For example,
  • WO 2014/063054 describes a large range of possible substituent groups (these are the groups -R c and -R F in this prior art). Thus, the compounds of the present case are not limited to those where -A- is not further substituted or is further substituted with methyl.
  • -R A is present, it is typically a substituent to a ring carbon atom.
  • Each group -R A is independently selected from -L AA -R M , halo, hydroxy (-OH), amino (-NH 2 ), thiol (-SH), cyano, nitro, and carboxy (-COOH), where: a covalent bond or is selected from * -C(0)-, * -S(0)-, * -S(0) 2 - * -N(H)C(0)-, *-N(H)S(0)-, * -N(R N )S(0)-, * -N(H)S(0) 2 -, * -N(R N )S(0) 2 -, * -N(H)-, and -N(R N )-, i-e alkyl, and the asterisk indicates the point of attachment to the cyclic group; selected from optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl and aryl.
  • each optional substituent may be selected from halo, hydroxy (-OH), amino (-NH 2 ), thiol (-SH), cyano (-CN), nitro, carboxy (-COOH), and phenyl, and where -R AA is cycloalkyl, heterocyclyl or aryl, the optional substituent is further selected from alkyl, such as Ci-e alkyl, such as methyl and ethyl.
  • the group -L ⁇ - is preferably a covalent bond.
  • the group -R AA is directly connected to the cyclic group -A-. This is preferred.
  • the group -R AA may be alkyl, such as Ci-e alkyl, such as methyl or ethyl.
  • the group -R AA may be alkenyl, such as C 2-6 alkenyl, such as ethenyl or propenyl.
  • the group -R AA may be alkynyl, such as C 2 -e alkynyl, such as ethynyl or propynyl.
  • alkyl, alkenyl or alkynyl group may be linear or branched.
  • the group -R AA may be cycloalkyl, such as C3-14 cycloalkyl, such as cyclopentyl and cyclohexyl.
  • the cycloalkyl group may be monocyclic, or may contain two or more fused rings, where each ring is a cycloalkyl ring.
  • a cycloalkyl group is nonaromatic.
  • a cycloalkyl ring may be unsaturated or partially saturated or fully saturated (but not aromatic).
  • the group -R AA may be heterocyclyl, such as C3-14 heterocyclyl, such as pyrrolinyl, piperidinyl, tetrahydrofuranyl and tetrahydropyranyl.
  • the heterocyclyl group may be monocyclic, or may contain two or more fused rings, where one ring is a heterocyclyl ring and the other rings may be cycloalkyl or heterocyclyl rings.
  • the heterocyclyl group is nonaromatic. Each ring in the heterocyclyl group may be unsaturated or partially saturated or fully saturated (but not aromatic).
  • the group -R AA may be aryl, such as carboaryl and heteroaryl.
  • the carboaryl may be Ce-14 carboaryl, such as phenyl and naphthyl.
  • the heteroaryl may be C5-14 heteroaryl, such as C5-10 heteroaryl, such as pyridinyl, pyrrolyl, furanyl and thiophenyl.
  • a halo group may be selected from fluoro, chloro, bromo and iodo, such as fluoro.
  • the group -R A is -R M or halo, such as -R A is alkyl or halo, such as methyl or fluoro. Most preferably, -R A is methyl or ethyl, such as most preferably methyl.
  • the group -L- is a link between the cyclic group -A- and the acceptor group -D.
  • the group -L- may be a covalent bond or alkylene.
  • the group -L- is a covalent bond.
  • WO 2014/063054 describes compounds where the group -A- may be connected to a Michael acceptor group via a linker that is a hydrocarbon chain (these are the groups -L- and -V- in this prior art).
  • the compounds of the present case are not limited to those where the cyclic group -A- connects directly to -D.
  • -L- is alkylene this may be Ci-e alkylene, such as C1-4 alkylene, such as methylene or ethylene.
  • An alkylene group is a saturated aliphatic group. The alkylene group may be linear or branched.
  • -L- is a covalent bond.
  • the cyclic group -A- connects directly to -D.
  • the group -D is an acceptor group, which is suitable for reaction with a nucleophilic group present within a polypeptide, such as a protein.
  • the acceptor group is preferably reactive with thiol functionality, which may be present within the side chain of a cysteine amino acid residue of a polypeptide.
  • the compounds of the invention are preferably reacted with a kinase, such as those described herein, including BMX, to form a complex of the compound with the kinase.
  • a kinase such as those described herein, including BMX
  • the compound is covalently linked to the kinase via the acceptor group and a site on the kinase.
  • the group -D is an amide connecting to an ethenyl group. It is known from the art that other acceptor group may be used at this position.
  • WO 2014/063054 describes compounds having a range of acceptor groups, (these are the groups -R D and -R G in this prior art).
  • the compounds of the present case are not limited to the use of an amide connecting to an ethenyl group at -D.
  • the acceptor group may contain an a,b-unsaturated carbonyl group or a,b-unsaturated thiocarbonyl group.
  • the acceptor group may be -X-M, where -X- is a covalent bond or -L M -, and -M is selected from alkenyl, alkynyl, heterocyclyl, alkyl substituted with cyano, and cyano.
  • the group -M may contain an unsaturated bound, such as a carbon-carbon double bond, for example where -M is alkenyl, or heterocyclyl. Preferably this group is provided a,b to the group -X-. This is particularly preferred where -X- contains a carbonyl group (-C(O)-).
  • the unsaturated bond may be a carbon-carbon triple bond, for example where -M is alkynyl.
  • the unsaturated bond may be a carbon-nitrogen triple bond, for example where a cyano group is present.
  • the group -L M - may be selected from * -C(0)-, * -S(0)-, * -S(0) 2 - * -N(H)C(0)-, * -N(R N )C(0)-, * -N(H)S(0)-, * -N(R N )S(0)-, * -N(H)S(0) 2 -, *-N(R N )S(0) 2 -, * -N(H)-, and -N(R N )-, where -R N is C1-6 alkyl, and the asterisk indicates the point of attachment to -L- (and where -L- is a covalent bond, this is the point of attachment to -A-).
  • -X- may be a covalent bond group, or -X- may be -L M - where -L M - is selected from * -C(0)-, * -S(0)-, and * -S(0) 2 -.
  • the group -L M - is typically * -C(0)-, * -S(0)-, * -S(0) 2 -, * -N(H)C(0)- or * -N(R N )C(0)-, such as * -N(H)C(0)- or * -N(R N )C(0)-, such as * -N(H)C(0)-.
  • the group -M comprises the reactive functionality for forming a covalent bond to a polypeptide, such as a kinase.
  • the reactive functionality is electrophilic, and more specifically, together with group -X-, it is a Michael acceptor group.
  • alkenyl this may be C 2-6 alkenyl.
  • the alkenyl group may contain one carbon- carbon double bond.
  • the double bond is conjugated with a carbonyl group present as -X- (thus, providing a a,b-unsaturated carbonyl group or a,b-unsaturated thiocarbonyl group).
  • -M is alkynyl this may be C 2-6 alkynyl.
  • the alkynyl group may contain one carbon- carbon triple bond.
  • the triple bond is conjugated with a carbonyl group present as -X-.
  • heterocyclyl this may be a saturated or partially unsaturated heterocyclyl.
  • the heterocyclyl may be a C3-7 heterocyclyl, such as a C3 or a C5 heterocyclyl.
  • a heterocyclyl group may be optionally substituted with carbonyl (-C(O)-) at an available ring carbon atom.
  • carbonyl preferably, it may be a substituent to a ring carbon atom that is a to a ring heteroatom, such as a ring nitrogen atom, for example to from an internal (endo) amide or imide.
  • the heterocyclyl is a C3 heterocyclyl it is preferably unsaturated.
  • a C 3 heterocyclyl may be selected from aziridinyl, oxiranyl and thiiranyl.
  • heterocyclyl is a C 5 heterocyclyl it is preferably partially saturated, and preferably contains a single carbon-carbon double bond.
  • -M is maleimidyl, which is connected to -X- through the ring nitrogen atom.
  • a maleimidyl group is a C5 nitrogen heterocyclyl group where each of the carbon ring atoms a to the ring nitrogen atom are substituted with carbonyl.
  • the group -M may be alkyl substituted with cyano.
  • the alkyl group may be C1-10 alkyl, such as C1 -6 alkyl, substituted with cyano.
  • the alkyl may be methyl or ethyl, such as methyl, substituted with cyano.
  • -M is selected from optionally substituted alkenyl, optionally substituted alkynyl, cyano, and alkyl substituted with cyano.
  • the group -D such as -L-D together, may be selected from -N(H)C(0)CHCH 2 ,
  • the group -D such as -L-D together, is preferably -N(H)C(0)CHCH 2 .
  • the group -CF 3 is not an acceptor group.
  • -D cannot be -CF 3 .
  • the group -R 7 is a substituent at the 7-position of the quinoline ring.
  • the compounds known in the art are substituted only at the 6-position.
  • the present inventors have shown that a range of different groups are tolerated at this position.
  • WO 2014/063054 describes compounds where the tricyclic core is substituted at the 6-position of the quinoline ring of that core.
  • the substituent that is present at the 6-position is typically an aromatic group connected to the quinoline ring, either directly or via a Ci-e hydrocarbon linker.
  • Such a substituent may be provided at the 7-position within the compounds of the invention.
  • -R 7 may contain aryl.
  • the presence of a 7-susbituent group may be associated with an improved biological activity compared with a related compounds having the same substituent at the 7-position.
  • alternative groups may be used at the 7-position, where such groups have never been described for use at the 6-position.
  • WO 2014/063054 describes substituent to the tricyclic core that contain an aryl group.
  • the inventors have shown that alternative groups may be used as substituents to the tricyclic core, for example including substituted cycloalkyl and heterocyclyl, amongst others.
  • the presence of such groups may be associated with a comparable or improved biological activity compared with those compounds substituted at the 6-position, or those compounds having an aryl group within the substituent to the tricyclic core.
  • the group -R 7 is -L 7A -L 7B -R 7A , where
  • -L 7A - is a covalent bond, or is selected from *-0-, *-S-, *-NH-, *-N(R N )-,
  • -L 7B - is a covalent bond or selected from C1-6 alkylene, C 2-6 alkenylene,
  • -R 7A is selected from optionally substituted cycloalkyl, heterocyclyl, and aryl, and when -L 7B - is a covalent bond, -R 7A is further selected from optionally substituted alkyl, alkenyl, alkynyl and heteroalkyl.
  • a group -R 7A may be optionally substituted with one or more groups -R s . Where two or more groups -R s are present, each -R s may be the same or different. These optional substituents are defined in detail below.
  • the group -R 7 contains a nitrogen atom.
  • -R 7A is heterocyclyl or aryl (for example, heteroaryl), or where the group -R s contain a nitrogen atom, for example where -R s includes a sulfonamide group.
  • the groups -L 7A - and -L 7B - are linkers that connect the quinoline ring of the core to the group -R 7A .
  • the group -R 7A may be connected directly to the quinoline ring.
  • each of -L 7A - and -L 7B - is a covalent bond.
  • each of -L 7A - and -L 7B - is a covalent bond.
  • -R 7 is -R 7A .
  • -R 7A is connected directly to the tricyclic ring.
  • -R 7 is -L 7B -R 7A , and preferably -L 7B - is C 2-6 alkenylene.
  • -R 7 is -L 7A -R 7A , and preferably -L 7A - is -NH- or -N(R N )-.
  • -R 7A is aryl it may be carboaryl or heteroaryl.
  • a carboaryl group may be Ce-u carboaryl, such as phenyl or naphthyl, and preferably phenyl.
  • a heteroaryl group may be C 5-14 heteroaryl, such as C 5-10 heteroaryl, such as C 5-6 heteroaryl.
  • An aryl group may be monocyclic, or may comprise a plurality of fused rings. Where a plurality of rings is present, the ring connected to -L 7B - is aromatic. The other rings are optionally aromatic. The other rings may be fully unsaturated or partially unsaturated. The other rings may be independently selected from aromatic, cycloalkyl and heterocyclyl rings.
  • -R 7A is optionally substituted aryl, such as optionally substituted phenyl, pyridinyl, pyrrolyl, oxazolyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, quinolinyl and isoquinolinyl.
  • aryl such as optionally substituted phenyl, pyridinyl, pyrrolyl, oxazolyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, quinolinyl and isoquinolinyl.
  • -R 7A is optionally substituted phenyl or pyridinyl, such as phenyl.
  • the phenyl or the pyridinyl is optionally substituted, such as optionally monosubstituted.
  • -R 7A When -R 7A is heterocyclyl it may be C 3-14 heterocyclyl.
  • a heterocyclyl has one or two ring heteroatoms, with each ring heteroatom selected from O, S and N(H).
  • the heterocyclyl may connect to -L 7B - via a ring carbon atom or a ring nitrogen atom, where present.
  • a heterocyclyl may be partially unsaturated (but not aromatic).
  • the heterocyclyl may be monocyclic, or may comprise a plurality of fused rings. Where a plurality of rings is present, the ring connected to -L 7B - is non-aromatic, and is preferably a fully saturated ring.
  • the other rings are optionally aromatic.
  • the other rings may be fully unsaturated, partially unsaturated or saturated.
  • the other rings may be independently selected from aromatic, cycloalkyl and heterocyclyl rings.
  • the heterocyclyl group may be selected from piperidinyl, piperazinyl, morpholinyl, and thiomorpholinyl, such as selected from piperidinyl and piperazinyl.
  • -R 7A When -R 7A is cycloalkyl it may be C 3-10 cycloalkyl, such as C 5-10 cycloalkyl.
  • the cycloalkyl may be monocyclic, or may comprise a plurality of fused rings.
  • a cycloalkyl group may be partially unsaturated (but not aromatic).
  • the ring connected to -L 7B - is non-aromatic, and is preferably a fully saturated ring.
  • the other rings are optionally aromatic.
  • the other rings may be fully unsaturated, partially unsaturated or saturated.
  • the other rings may be independently selected from aromatic, cycloalkyl and heterocyclyl rings.
  • a cycloalkyl group may be selected from cyclopentylene (Cs), cyclohexylene (Ce);
  • tetralinylene and decalinylene such as cyclohexylene.
  • -R 7A is alkyl it may be Ci-e alkyl, such as C1-4 alkyl, such methyl or ethyl.
  • the alkyl group may be linear or branched. The alkyl may be optionally substituted.
  • alkenyl it may be C 2-6 alkenyl, such as C 2 -4 alkenyl, such ethenyl.
  • the alkenyl group may be linear or branched.
  • the alkenyl may be optionally substituted.
  • alkynyl When -R 7A is alkynyl it may be C 2-6 alkynyl, such as C 2 -4 alkynyl, such ethynyl.
  • the alkynyl group may be linear or branched.
  • the alkynyl may be optionally substituted.
  • heteroalkyl When -R 7A is heteroalkyl it may be C 2-6 heteroalkyl, such as C3-6 heteroalkyl.
  • the alkynyl group may be linear or branched.
  • a heteroalkyl group is an alkyl group where one or two carbon atoms is replaced with a heteroatom selected from O, S and N(H). The heteroatom does not replace a carbon atom at the terminal of the alkyl group.
  • the heteroalkyl group may be connected via a heteroatom, or alternatively it may be connected via a carbon atom.
  • the group -R 7A is preferably selected from optionally substituted aryl and heterocyclyl.
  • the group -R 6 is a substituent at the 6-position of the quinoline ring.
  • the substituent does not contain an aromatic group connected to the quinoline ring, or the 6-substituent does not contain an aromatic group connected to the quinoline ring via a Ci-e hydrocarbon linker.
  • the compounds known in the art are substituted at the 6-position with an aromatic group, which is directly linked to the quinoline ring, or is connected via a Ci-e hydrocarbon linker. See, for example, the compounds described in WO 2014/063054.
  • the compounds are not for use in the treatment of proliferative diseases, such as cancer, and they are not disclosed for use as binders to any kinase. Rather, the compounds are used for their antiviral activity.
  • the group -R 6 is -
  • -L 6A - is a covalent bond or is selected from *-0-, *-S-, *-NH-, *-N(R N )-, *-C(0)-, * -C(0)NH-, * -C(0)N(R N )-, * -NHC(0)-, * -N(R N )C(0)-, * -S(0) 2 NH-, * -S(0) 2 N(R N )-, * -NHS(0) 2 - and *-N(R N )S(0) 2 -, where -R N is Ci-e alkyl and the asterisk indicates the point of attachment to the quinoline;
  • -L 6B - is selected from a covalent bond or selected from Ci-e alkylene
  • the group -R 6 may not be morpholinyl, such as morpholin-4-yl (that is, a morpholinyl group connected to the quinolone ring via the morpholine ring nitrogen). Such a limitation may be limited to unsubstituted morpholinyl groups only.
  • the group -R 6 may not be optionally substituted piperazinyl, such as optionally substituted piperazin-1-yl. More specifically, -R 6 may not be 4-phenylpiperazin-1-yl.
  • a group -R 6A may be optionally substituted with one or more groups -R s . Where two or more groups -R s are present, each -R s may be the same or different. These optional substituents are defined in detail below.
  • the group -R 6 contains a nitrogen atom.
  • -R 6A is heterocyclyl, or where the group -R s contain a nitrogen atom, for example where -R s includes a sulfonamide group.
  • the groups -L 6A - and -L 6B - are linkers that connect the quinoline ring to the group -R 6A .
  • the group -R 6A may be connected directly to the quinoline ring.
  • each of -L 6A - and -L 6B - is a covalent bond.
  • each of -L 6A - and -L 6B - is a covalent bond.
  • -R 6 is -R 6A .
  • -R 6A is connected directly to the tricyclic ring.
  • -R 6 is -L 6B -R 6A , and preferably -L 6B - is C alkenylene.
  • -R 6 is -L 6A -R 6A , and preferably -L 6A - is -NH- or -N(R N )-.
  • -R 6A When -R 6A is heterocyclyl it may be C3-14 heterocyclyl, such as C5-7 heterocyclyl, such as C5-6 heterocyclyl.
  • a heterocyclyl has one or two ring heteroatoms, with each ring heteroatom selected from O, S and N(H).
  • the heterocyclyl may connect to -L 7B - via a ring carbon atom or a ring nitrogen atom, where present.
  • a heterocyclyl may be partially unsaturated (but not aromatic).
  • the heterocyclyl may be monocyclic, or may comprise a plurality of fused rings. Where a plurality of rings is present, the ring connected to -L 6B - is non-aromatic, and is preferably a fully saturated ring.
  • the other rings are optionally aromatic.
  • the other rings may be fully unsaturated, partially unsaturated or saturated.
  • the other rings may be independently selected from aromatic, cycloalkyl and heterocyclyl rings.
  • the heterocyclyl group may be selected from piperidinyl, piperazinyl, morpholinyl, and thiomorpholinyl, such as piperidinyl, piperazinyl, and thiomorpholinyl, such as selected from piperidinyl and piperazinyl or selected from piperidinyl and thiomorpholinyl.
  • the heterocyclyl group may not be morpholinyl, for example where each of -L 6A - and -L 6B - is a covalent bond.
  • the heterocyclyl group may not be piperazinyl, for example where each of -L 6A - and -L 6B - is a covalent bond.
  • -R 6A When -R 6A is cycloalkyl it may C 3-10 cycloalkyl, such as C 5-10 cycloalkyl.
  • the cycloalkyl may be monocyclic, or may comprise a plurality of fused rings.
  • a cycloalkyl group may be partially unsaturated (but not aromatic).
  • the ring connected to -L 6B - is non-aromatic, and is preferably a fully saturated ring.
  • the other rings are optionally aromatic.
  • the other rings may be fully unsaturated, partially unsaturated or saturated.
  • the other rings may be independently selected from aromatic, cycloalkyl and heterocyclyl rings.
  • a cycloalkyl group may be selected from cyclopentylene (C 5 ), cyclohexylene (Ob);
  • tetralinylene and decalinylene such as cyclohexylene.
  • -R 6A When -R 6A is alkyl it may be Ci-e alkyl, such as C 1-4 alkyl, such methyl or ethyl.
  • the alkyl group may be linear or branched.
  • the alkyl may be optionally substituted.
  • alkenyl it may be C 2-6 alkenyl, such as C 2-4 alkenyl, such ethenyl.
  • the alkenyl group may be linear or branched.
  • the alkenyl may be optionally substituted.
  • alkynyl When -R 6A is alkynyl it may be C 2-6 alkynyl, such as C 2-4 alkynyl, such ethynyl.
  • the alkynyl group may be linear or branched.
  • the alkynyl may be optionally substituted.
  • heteroalkyl When -R 6A is heteroalkyl it may be C 2-6 heteroalkyl, such as C 3-6 heteroalkyl.
  • the heteroalkyl group may be linear or branched.
  • a heteroalkyl group is an alkyl group where one or two carbon atoms is replaced with a heteroatom selected from O, S and N(H). The heteroatom does not replace a carbon atom at the terminal of the alkyl group.
  • the heteroalkyl group may be connected via a heteroatom, or alternatively it may be connected via a carbon atom.
  • the group -R 6A is preferably optionally substituted heterocyclyl.
  • the group -R 6 contains no aromatic functional group, for example, the group -R 6 does not contain a phenyl group.
  • the group -R s may be provided as a substituent to the group -R 7A or the group -R 6A . This substituent is optionally present. Typically each of -R 7A and -R 6A is unsubstituted, or is monosubstituted with -R s . In other embodiments each of -R 7A and -R 6A is provided with two or more substituents -R s .
  • the group -R s may be a substituent to a carbon atom within the group -R 6A or -R 7A .
  • the group -R s is -R sc .
  • the group -R s may be a substituent to a nitrogen atom within the group -R 6A or -R 7A .
  • the group -R s is -R SN .
  • each -R sc is independently selected from -L sc -R ss , halo, hydroxy (-OH), amino (-NH2), thiol (-SH), cyano, nitro, and carboxy (-COOH), where:
  • -L sc - is a covalent bond or is selected from * -C(0)-, * -S(0)-, * -S(0) 2 - * -N(H)C(0)-, * -N(R N )C(0)-, * -N(H)S(0)-, * -N(R N )S(0)-, * -N(H)S(0) 2 -, * -N(R N )S(0) 2 -, * -N(H)-, and -N(R N )-, where -R N is Ci-e alkyl, and the asterisk indicates the point of attachment to R 6A or -R 7A ; and -R ss is selected from optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl and aryl.
  • -L sc - is a covalent bond or is selected from *-N(H)S(0)-, *-N(R N )S(0)-, * -N(H)S(0) 2 -, *-N(R N )S(0) 2 -, such as -L sc - is a covalent bond or * -N(R N )S(0) 2 -.
  • each -R SN is independently selected from -L SN -R SS , where:
  • -L SN - is a covalent bond or is selected from * -C(0)-, * -S(0)-, * -S(0) 2 - , and the asterisk indicates the point of attachment to R 6A or -R 7A ;
  • -R ss is selected from optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl and aryl.
  • -L sc - is a covalent bond or is selected from * -S(0)-, * -S(0) 2 -, such as -L sc - is a covalent bond or * -S(0) 2 -.
  • -R ss is alkyl it may be Ci-e alkyl, such as C1.4 alkyl, such methyl or ethyl.
  • the alkyl group may be linear or branched.
  • the alkyl may be optionally substituted.
  • alkenyl When -R ss is alkenyl it may be C2-6 alkenyl, such as C2-4 alkenyl, such ethenyl.
  • the alkenyl group may be linear or branched.
  • the alkenyl may be optionally substituted.
  • alkynyl When -R ss is alkynyl it may be C2-6 alkynyl, such as C2-4 alkynyl, such ethynyl.
  • the alkynyl group may be linear or branched.
  • the alkynyl may be optionally substituted.
  • cycloalkyl When -R ss is cycloalkyl it may C3-10 cycloalkyl, such as C5-10 cycloalkyl.
  • the cycloalkyl may be monocyclic, or may comprise a plurality of fused rings.
  • a cycloalkyl group may be partially unsaturated (but not aromatic).
  • the ring connected to -L SN - or -L sc - is non-aromatic, and is preferably a fully saturated ring.
  • the other rings are optionally aromatic.
  • the other rings may be fully unsaturated, partially unsaturated or saturated.
  • the other rings may be independently selected from aromatic, cycloalkyl and heterocyclyl rings.
  • a cycloalkyl group may be selected from cyclopentylene (C 5 ), cyclohexylene (Ob);
  • tetralinylene and decalinylene such as cyclohexylene.
  • a heterocyclyl has one or two ring heteroatoms, with each ring heteroatom selected from O, S and N(H).
  • the heterocyclyl may connect to -L SN - or -L sc - via a ring carbon atom or a ring nitrogen atom, where present.
  • a heterocyclyl may be partially unsaturated (but not aromatic).
  • the heterocyclyl may be monocyclic, or may comprise a plurality of fused rings. Where a plurality of rings is present, the ring connected to -L SN - or -L sc - is non-aromatic, and is preferably a fully saturated ring.
  • the other rings are optionally aromatic.
  • the other rings may be fully unsaturated, partially unsaturated or saturated.
  • the other rings may be independently selected from aromatic, cycloalkyl and heterocyclyl rings.
  • the heterocyclyl group may be selected from piperidinyl, piperazinyl, morpholinyl, and thiomorpholinyl.
  • -R ss is aryl it may be carboaryl or heteroaryl.
  • a carboaryl group may be Ce-u carboaryl, such as phenyl or naphthyl, and preferably phenyl.
  • a heteroaryl group may be C 5-14 heteroaryl, such as C 5-10 heteroaryl, such as C 5-6 heteroaryl.
  • An aryl group may be monocyclic, or may comprise a plurality of fused rings. Where a plurality of rings is present, the ring connected to -L SN - or -L sc - is aromatic. The other rings are optionally aromatic. The other rings may be fully unsaturated or partially unsaturated. The other rings may be independently selected from aromatic, cycloalkyl and heterocyclyl rings.
  • -R ss is optionally substituted aryl, such as optionally substituted phenyl, pyridinyl, pyrrolyl, oxazolyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, quinolinyl and isoquinolinyl.
  • -R ss is alkyl
  • each optional substituent may be selected from the group consisting of halo (such as -F, -Cl, -Br, and -I), hydroxy (-OH), amino (-NH 2 ), thiol (-SH), cyano (-CN), nitro, carboxy (-COOH), and phenyl, and where -R ss is cycloalkyl, heterocyclyl or aryl, the optional substituent is further selected from alkyl, such as Ci-e alkyl, such as methyl and ethyl.
  • the compounds of formula (I) may be a compound as set out below:
  • -R A , -R 7 and -M have the same meanings as given above.
  • the phenylene group attached to the pyridone nitrogen is monosubstituted with -R A .
  • This group may be provided at any one of the 2-, 4-, 5- or 6-positions, and preferably at the 4-position.
  • the compounds of formula (I) may be a compound as set out below:
  • the compounds of formula (I) may be a compound as set out below:
  • the compounds of formula (II) may be a compound as set out below:
  • -R A , -R 6 and -M have the same meanings as given above.
  • the phenylene group attached to the pyridone nitrogen is monosubstituted with -R A .
  • This group may be provided at any one of the 2-, 4-, 5- or 6-positions, and preferably at the 4-position.
  • the compounds of formula (II) may be a compound as set out below:
  • the compounds of formula (II) may be a compound as set out below:
  • the present invention also provides a compound of formula (I) or a compound of formula (II) covalently bound to a polypeptide. This combination may be referred to as a complex of the compound with the polypeptide.
  • the polypeptide typically contains a threonine amino acid residue, and the compound of formula (I) or (II) is bound to the polypeptide through the side-chain functionality of this threonine residue.
  • a complex may be formed by contacting a compound of formula (I) or (II) with a polypeptide.
  • the compounds of formula (I) and (II) are provided with an acceptor group, such as a Michael acceptor group, that is suitable for reaction with a side chain functionality of an amino residue of the polypeptide, such as a thiol functionality of a cysteine residue.
  • the polypeptide is a kinase.
  • the kinase may be selected from a kinase family selected from TEC, EGFR. JAK, Src, FAK, PI3K, mTOR, Liver Kinase B1 , Pkb, PAK1 , TAM, Abl and PDPK1.
  • a TEC kinase family member may be selected from the group consisting of BMX, BTK, ITK, TEC and TXK.
  • An EGFR kinase family member may be selected from the group consisting of EGFR, ERBB2, ERBB3 and ERBB4.
  • a JAK kinase family member may be selected from the group consisting of JAK1 , JAK2, JAK3 and TYK2.
  • a Src kinase family member may be selected from the group consisting of FYN, SRC, YES1 , BLK, FGR, LCK, HCK, and LYN.
  • a FAK kinase family member may be PTK2.
  • a PI3k kinase family member may be selected from the group consisting of PIK3CA,
  • PIK3CP PIK3CP
  • PIK3Cy PIK3C5.
  • a mTOR kinase family member may be mTOR.
  • a Liver Kinase B1 kinase family member may be Liver Kinase B1.
  • a Pkb kinase family member may be selected from the group consisting of ATK1 , ATK2 and ATK3.
  • a PAK1 kinase family member may be PAK1.
  • a TAM kinase family member may be selected from AXL and MERTK.
  • a Abl kinase family member may be Abl 1.
  • a PDPK1 kinase family member may be PDPK1.
  • the kinase is a TEC kinase family, and most preferably the kinase is BMK or BTK, such as BMX.
  • the kinase may be a human kinase.
  • the polypeptide is an enzyme with kinase activity, where the polypeptide has an amino acid sequence as set out in SEQ ID Nos.: 1 to 6, or a variant thereof.
  • the polypeptide may comprise a polypeptide having at least 35%, 45%, 55%, 65%, 75%, 85%, 95%, 98%, 99% or 100% identity to any one of SEQ ID Nos.: 1 to 6, such as SEQ ID No.: 1.
  • the kinase may comprise a TH domain, and typically comprise a cysteine residue in the pocket of the active site.
  • the kinase may be BMX, such as a BMX comprising a polypeptide having the amino acid sequence as set out in SEQ ID No.: 1.
  • a compound of formula (I) or (II) may be bound to the side chain of the Cys 496 residue of BMX.
  • the compound may be bound to a cysteine residue that corresponds to the Cys 496 residue of BMX.
  • Amino acid sequence identity and similarity may be measured using standard bioinformatics software tools, such as the freely available EMBOSS, or BLAST, software tools. Default parameters are generally used.
  • EMBOSS Needle pairwise sequence alignment can be used to determine amino acid sequence identity.
  • Use of GAP may be preferred but other algorithms may be used, e.g.
  • BLAST or TBLASTN which use the method of Altschul et al. (1990) J. Mol. Biol. 215: 405- 410
  • FASTA which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444- 2448
  • Smith-Waterman algorithm Smith and Waterman (1981) J. Mol Biol. 147: 195- 197
  • a % amino acid sequence identity value is determined by the number of matching identical residues as determined by WU-BLAST-2, divided by the total number of residues of the reference sequence (gaps introduced by WU- BLAST-2 into the reference sequence to maximize the alignment score being ignored), multiplied by 100.
  • Percent (%) amino acid sequence alignment coverage with respect to a reference sequence is defined as the percentage of amino acid residues in the candidate sequence in
  • a variant polypeptide may be a truncated polypeptide. Any truncation may be used as long as the truncated polypeptide still has kinase activity. Truncations may remove one or more residues from the N- and/or C-terminus of the polypeptide, which residues are non-essential for kinase activity. Appropriate truncations may be routinely identified by systematic truncation of sequences of varying length from the N- and/or C-terminus.
  • a variant polypeptide comprise one or more additional amino acids.
  • a variant polypeptide may comprise an affinity tag for purifying the variant polypeptide, such as a poly-histidine tag, a T7 tag or a GST tag.
  • An affinity tag may be located at the N- or C- terminus.
  • the variant polypeptide may further comprise a leader sequence at the N-terminus.
  • the leader sequence may be useful for directing secretion and/or intracellular targeting of the polypeptide in a recombinant expression system.
  • Leader sequences are also known as signal peptides and are well known in the art.
  • the polypeptide may further comprise a label such as a fluorescent label.
  • Amino acid substitutions may be conservative amino acid substitutions, in which an amino acid of a given sequence is substituted by an amino acid having similar characteristics. For example, where a hydrophobic amino acid (e.g. Leu) is substituted by another hydrophobic amino acid (e.g. lie). Amino acids and conservative substitutions are shown in the table below. A conservative substitution may be defined as a substitution within an amino acid class and/or a substitution that scores positive in the BLOSUM62 matrix.
  • salts of compound of formula (I) and (II) include all pharmaceutically acceptable salts, such as, without limitation, acid addition salts of strong mineral acids such as HCI and HBr salts and addition salts of strong organic acids such as a methanesulfonic acid salt. Further examples of salts include sulphates and acetates such as trifluoroacetate or trichloroacetate.
  • a compound of formula (I) or (II) can also be formulated as prodrug.
  • Prodrugs can include a compound herein described in which one or more amino groups are protected with a group which can be cleaved in vivo, to liberate the biologically active compound.
  • a compound of formula (I) or (II) is provided as a prodrug.
  • a reference to a compound of formula (I) or (II), or any other compound described herein, is also a reference to a solvate of that compound.
  • solvates include hydrates.
  • a compound of formula (I) or (II), or any other compound described herein includes a compound where an atom is replaced by a naturally occurring or non-naturally occurring isotope.
  • the isotope is a stable isotope.
  • a compound described here includes, for example deuterium containing compounds and the like.
  • H may be in any isotopic form, including 1 H, 2 H (D), and 3 H (T);
  • C may be in any isotopic form, including 12 C, 13 C, and 14 C;
  • O may be in any isotopic form, including 16 0 and 18 0; and the like.
  • Certain compounds of formula (I) or (II), or any other compound described herein, may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate- forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and b-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as“isomers” (or
  • isomers are structural (or constitutional) isomers (i.e. , isomers which differ in the connections between atoms rather than merely by the position of atoms in space).
  • a reference to a methoxy group, -OCH3 is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH2OH.
  • a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta- chlorophenyl.
  • Ci- 6 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para- methoxyphenyl).
  • a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof.
  • Methods for the preparation e.g., asymmetric synthesis
  • separation e.g., fractional crystallisation and
  • One aspect of the present invention pertains to compounds in substantially purified form and/or in a form substantially free from contaminants.
  • the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight.
  • substantially purified form refers to the compound in any
  • the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds.
  • the substantially purified form refers to one stereoisomer, e.g., optically pure stereoisomer.
  • the substantially purified form refers to a mixture of enantiomers.
  • the substantially purified form refers to an equimolar mixture of enantiomers (i.e. , a racemic mixture, a racemate).
  • the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer.
  • the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1 % by weight.
  • the contaminants refer to other compounds, that is, other than
  • the contaminants refer to other compounds and other stereoisomers. In one embodiment, the contaminants refer to other compounds and the other enantiomer.
  • the substantially purified form is at least 60% optically pure (i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer), e.g., at least 70% optically pure, e.g., at least 80% optically pure, e.g., at least 90% optically pure, e.g., at least 95% optically pure, e.g., at least 97% optically pure, e.g., at least 98% optically pure, e.g., at least 99% optically pure.
  • 60% optically pure i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer
  • at least 70% optically pure e.g., at least 80% optically pure, e.g., at least 90% optically pure, e
  • the compounds of formula (I) or (II), or pharmaceutical formulations containing these compounds are suitable for use in methods of treatment and prophylaxis.
  • the compounds may be administered to a subject in need thereof.
  • the compounds of formula (I) or (II) are for use in a method of treatment of the human or animal body by therapy.
  • a compound of formula (I) or (II) may be administered to a mammalian subject, such as a human, in order to treat a proliferative disease, such as cancer.
  • Another aspect of the present invention pertains to use of a compound of formula (I) or (II) in the manufacture of a medicament for use in treatment.
  • the medicament comprises a compound of formula (I) or (II).
  • the compounds of the present case may be useful for the treatment of a proliferative disease, such as cancer.
  • the cancer may be selected from breast, prostate, colon and cervical cancers, leukaemia, myeloma and non-Hodgkin's lymphoma.
  • the compounds of the invention may be used to treat an autoimmune disease.
  • the autoimmune disease may be for example rheumatoid arthritis or lupus (see, for example. Honignerg et ai, 2010, Xia et ai, 2010, Chalmers et ai, 2015 and Rankin et ai, 2013).
  • the compounds of the invention may be used to treat a disease associated with kinase activity, such as elevated kinase activity.
  • treatment pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the condition, amelioration of the condition, and cure of the condition.
  • Treatment as a prophylactic measure i.e., prophylaxis
  • prophylaxis is also included.
  • treatment use with patients who have not yet developed the condition, but who are at risk of developing the condition.
  • therapeutically-effective amount refers to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
  • treatment includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I) or (II) together with a pharmaceutically acceptable carrier.
  • compositions, preparation, medicament comprising at least one compound of formula (I) or (II), as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to,
  • compositions may further comprise other active agents, for example, other therapeutic or prophylactic agents.
  • the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition
  • a pharmaceutical composition comprising admixing at least one compound of formula (I) or (II), as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound.
  • the composition optionally further comprises the second active agent in a predetermined amount.
  • ingredients, materials, compositions, dosage forms, etc. which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Each carrier, diluent, excipient, etc. must also be“acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 2005.
  • the formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the compound of formula (I) or (II) with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.
  • carriers e.g., liquid carriers, finely divided solid carrier, etc.
  • Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.
  • solutions e.g., aqueous, non-aqueous
  • suspensions e.g., aqueous, non-aqueous
  • Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir.
  • Formulations suitable for oral administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses.
  • Formulations suitable for buccal administration include mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
  • Losenges typically comprise the compound in a flavoured basis, usually sucrose and acacia or tragacanth.
  • Pastilles typically comprise the compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia.
  • Mouthwashes typically comprise the compound in a suitable liquid carrier.
  • Formulations suitable for sublingual administration include tablets, losenges, pastilles, capsules, and pills.
  • Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in- water, water-in-oil), mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
  • solutions e.g., aqueous, non-aqueous
  • suspensions e.g., aqueous, non-aqueous
  • emulsions e.g., oil-in- water, water-in-oil
  • mouthwashes e.g., losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
  • Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.
  • solutions e.g., aqueous, non-aqueous
  • suspensions e.g., aqueous, non-aqueous
  • emulsions e.g., oil-in-water, water-in-oil
  • suppositories e.g., pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.
  • Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.
  • Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients.
  • Ointments are typically prepared from the compound and a paraffinic or a water-miscible ointment base.
  • Emulsions are typically prepared from the compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
  • an emulsifier also known as an emulgent
  • a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat.
  • emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
  • Formulations suitable for intranasal administration, where the carrier is a liquid include, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the compound.
  • a dry powder delivery may be used as an alternative to nebulised aerosols.
  • Formulations suitable for intranasal administration, where the carrier is a solid include, for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e. , by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Formulations suitable for pulmonary administration include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro- tetrafluoroethane, carbon dioxide, or other suitable gases.
  • a formulaton for pulmonary administration may be formulated for administration from a nebuliser or a dry powder inhaler.
  • the formulation may be provided with carriers or liposomes to provide a suitable particle size to reach the appropriate parts of the lung, to aid delivery of an appropriate does to enhance retention in the lung tissue.
  • Formulations suitable for ocular administration include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound.
  • Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema.
  • a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate.
  • Formulations suitable for parenteral administration include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other micro particulate).
  • Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient.
  • excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.
  • Suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
  • concentration of the compound in the liquid is from about 1 ng/mL to about 100 pg/mL, for example from about 10 ng/mL to about 10 pg/mL, for example from about 10 ng/mL to about 1 pg/mL.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • kits comprising (a) a compound of formula (I) or (II), or a composition comprising a compound as defined in any one of formula (I) or (II), e.g., preferably provided in a suitable container and/or with suitable packaging; and
  • instructions for use e.g., written instructions on how to administer the compound or composition.
  • the written instructions may also include a list of indications for which the compound of formula (I) or (II) is a suitable treatment.
  • the kit further comprises (c) a second active agent, or a composition comprising the second active agent.
  • the written instructions may also include a list of indications for which the second active agent, together with the compound of formula (I) or (II), is suitable for treatment.
  • a compound of formula (I) or (II), a second agent, or a pharmaceutical composition comprising the compound of formula (I) or (II), may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e. , at the site of desired action).
  • Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary);
  • parenteral for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously, intracranially or intramuscularly.
  • the subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g
  • the subject/patient is a human.
  • a non-human mammal may be a rodent.
  • Rodents include rats, mice, guinea pigs, chinchillas and other similarly-sized small rodents used in laboratory research.
  • the present invention provides a method of treating a cell or a population of cells with a compound of formula (I) or a compound of formula (II), the method comprising the step of contacting a cell or cell population with a compound of formula (I) or a compound of formula (II).
  • the method may be performed in vitro or in vivo.
  • a cell or cell population may be obtained from a subject, such as a subject described herein.
  • a cell may be treated to limit or prevent its proliferation.
  • the cell may be a proliferative cell, such as cancer cell.
  • the cancer cell may be selected from breast, prostate, colon and cervical cancer cells.
  • cancer cells for treatment include prostate cancer cells, such as the LCNCaP, PC-3 and DU-145 and 22RV-1 cell lines exemplified herein, and further selected from chronic lymphocytic leukemia cell lines, JVM-3, M EC-2, MO1043 and WaC3CD5 cell lines.
  • prostate cancer cells such as the LCNCaP, PC-3 and DU-145 and 22RV-1 cell lines exemplified herein, and further selected from chronic lymphocytic leukemia cell lines, JVM-3, M EC-2, MO1043 and WaC3CD5 cell lines.
  • the method of treating a cell or a cell population may include the step of determining the proliferation of a cell or a cell population.
  • the methods may be used to determine the suitability of a compound for use in methods of treatment.
  • a compound of formula (I) may be administered in conjunction with a second agent.
  • Administration may be simultaneous, separate or sequential.
  • “separate” administration it is meant that a compound of formula (I) and a second agent are administered to a subject by two different routes of administration which occur at the same time. This may occur for example where one agent is administered by infusion and the other is given orally during the course of the infusion.
  • a sequential dose will occur such that the second agent is administered within 48 hours of administration of the compound of formula (I), preferably within 24 hours, such as within 12, 6, 4, 2 or 1 hour(s) of the first agent.
  • the active agent may be administered first, followed by the compound of formula (I).
  • the order and timing of the administration of the compound and second agent in the combination treatment will depend upon the pharmacokinetic properties of each.
  • the amount of the compound of formula (I) to be administered to a subject will ultimately depend upon the nature of the subject and the disease to be treated. Likewise, the amount of the active agent to be administered to a subject will ultimately depend upon the nature of the subject and the disease to be treated.
  • the second agent may be a substance which is a kinase inhibitor, for example a PI3K or AKT inhibitor.
  • the second agent may be LY294002 (CAS number: 154447-36- 6) or AKT1/2 kinase inhibitor.
  • the second agent may be an androgen receptor inhibitor, for example Flutamide (also known under trade name Eulexin).
  • the second agent may also act as an anti-cancer agent, such as one used to treat prostate cancer.
  • HRMS High resolution mass spectrometry
  • a compound of the invention includes compounds 24-27, where a group -R 7 is present. These are compounds of formula (I).
  • a compound of the invention includes compounds 20-23, where a group -R 6 is present. These are compounds of formula (II).
  • Compound 28 is provided as a further reference example.
  • Compound 29 is an intermediate compound useful for the preparation of the compounds of formula (II).
  • intermediates 7 were obtained via Suzuki cross-coupling with the corresponding boronic esters or acids or Buchwald-Hartwig aminations.
  • the amination procedure was not straightforward, and attempts were made with Pd2(dba)3 as palladium source, while screening K2CO3 or CS2CO3 as bases, together with BINAP, XPhos or tBuXPhos as phosphines.
  • successful reactions were obtained employing Pd(OAc)2, CS2CO3 and RBI NAP in dioxane at 90°C overnight. Reduction of the nitro group into amine was not performed following the literature procedure (SnCh) (Liu et ai ACS Chem. Biol. 2013).
  • a Suzuki coupling between intermediate 3a and the boronic ester was performed under the described conditions (see conditions g) in
  • 6-bromo-4-hydroxy-3-quinolinecarboxylate (13.4 g; 45.25 mmol) was suspended in SOCI2 (130 ml_; 1.792 mol) and the mixture heated to 80°C. After 5 h, a clear yellow solution was obtained. The solvent was evaporated and the solid co-evaporated with DCM (5 x) to remove residual HCI. It was dried in vacuum to afford the title compound as a light-yellow solid (14.4 g; 100% yield).
  • CiyHisBrNsCU 404.0240; found: 404.0244.
  • the alcohol 4a (2.34 g; 6.029 mmol) was suspended in DCM (150 ml_) and the mixture cooled to 0°C.
  • DMP (3.83 g; 9.041 mmol; 1.5 equiv.) was added portionwise and the reaction warmed to r.t. After 2 h, TLC analysis (5% MeOH in DCM) showed that the reaction was completed.
  • the solution was cooled to 0°C, and NaOH (1 M) was slowly added.
  • the mixture was stirred for 15 min at r.t.. H 2 0 was added and the phases were separated.
  • the aqueous phase was further extracted with DCM (3 x).
  • the combined organics were washed with brine, dried over MgSCU and taken to dryness to afford the title compound as a yellow solid (1.75 g; 75% yield).
  • CiyHisBrNsOs 386.0135; found: 386.0142.
  • CDCIs d 193.4, 154.9150.1, 149.7, 149.1, 142.0, 136.0, 132.2, 130.6, 128.8, 128.6, 120.8, 119.2, 119.1, 118.0, 113.9.
  • the aldehyde 5a (1.74 g; 4.505 mmol), triethyl phosphonoacetate (894 pl_; 4.505 mmol) and K2CO3 (1.87 g; 13.516 mmol; 3 equiv.) were mixed in dry EtOH (30 ml_) in a sealed tube under Argon. The mixture was heated to 100°C overnight. After 16 h, the reaction was cooled to r.t. and the solvent evaporated. The crude was partitioned between H2O and EtOAc. The aqueous phase was further extracted with EtOAc (3 x) and the combined organics washed with brine, dried over MgSCU and taken to dryness to afford the title compound as a dark brown solid (1.62 g; 88% yield).
  • the removal of the sulfonamide aromatic ring rendered compounds with less lipophilic character and also increased water solubility. More specifically, analogues 20-23 showed that the introduction of secondary cyclic amines is well tolerated and able to reduce cLogP up to 0.7 units and increase LogS by 1.6 units, when introduced in the 7-position of the quinolone ring.
  • BMX-IN-1 does not have an optimal physicochemical profile
  • the analogues could have limited membrane permeability.
  • Cell membrane permeability is of utmost importance for any drug molecule, even more if a molecule is targeting cytoplasmic proteins.
  • PAMPA parallel artificial membrane permeability assay
  • the PAMPA EvolutionTM instrument was used to determine permeability and we observed that the vast majority of the analogues have a high permeability (Table 1).
  • solubilizing motifs introduced in the sulfonamide region led to an increase in effective permeability.
  • the increased permeability may be mostly due to conformational aspects, such as intramolecular hydrogen bonding more than cLogP that does not consider three- dimensional conformation.
  • Dynamic light scattering (Zetasizer Nano S, Malvern, UK) was used to determine compound colloidal aggregation. The particle sizes were measured at 25°C. A 10 mM stock solution of test compound was prepared in DMSO, following dilution with deionized and filtered water to obtain an analyte solution of 10 mM (0.1% DMSO). Colloidal aggregation was measured through sequential dilutions at 10 pM, 1 pM and 0.1 pM.
  • Table 1 In silico cLogP and LogS calculation and in vitro artificial membrane permeability (PAM PA) and colloidal aggregation (DLS) determination.
  • the PAMPA EvolutionTM instrument was used to determine permeability, at Pion Inc.
  • a sandwich is formed such that each composite well is divided into two chambers, separated by a 125 pm thick microfilter disc (0.45 pm pores), coated with Pion GIT-0 phospholipid mixture.
  • the effective permeability, Pe (x 10 6 cm/sec), of each compound was measured at pH 6.8 in the donor compartment using low-binding, low UV Prisma buffer.
  • the drug-free acceptor compartment was filled with acceptor sink buffer containing a scavenger at the start of the test.
  • the proprietary scavenger mimics serum proteins and blood circulation, thus creating sink conditions.
  • Aqueous solutions of studied compounds are prepared by diluting and thoroughly mixing 3 pL of DMSO stock in 600 pL of Prisma HT buffer. Final concentration of organic solvent (DMSO) in aqueous buffer is £ 0.5% (v/v).
  • the reference solution is identical to the donor at time zero, so that any surface adsorption effects from the plastic ware is compensated.
  • the PAMPA sandwich was assembled and allowed to incubate for about 15 hours. The solutions in the donor compartment were un stirred within duration of the experiment. Thus, the thickness of the aqueous boundary layer expected to be about 1 ,000 pm.
  • the sandwich was then separated, and both the donor and receiver compartments were assayed for the amount of drug present by comparison with the UV spectrum obtained from reference standards. Mass balance was used to determine the amount of material remaining in the membrane filter and on the plastic (%R). All values are reported as the average of quadruplicates.
  • cLog P is a consensus value obtained as the arithmetic mean of five freely available predictive models (XLOGP3 (Cheng et aL), WLOGP (Wildman et aL), MLOGP (Moriguchi et al. Moriguchi et ai), SILICOS-IT and iLOGP (Daina et at.) and LogS is the arithmetic mean of two topological methods (ESOL model: Delaney et ai.. ⁇ AN et a!
  • PAMPA Pe is effective permeability (x 10 6 cm/sec) measured directly from assay at pH 6.8 and %R is membrane retention. All values are reported as the average of quadruplicates. Und label refers to compounds with extremely low solubility for which UV limits were below the detection limits therefore considered undetected. Compounds were labelled as high permeability (green), medium permeability (orange) or low permeability (red). DLS is measured at 10 pM, 1 pM and 100 nM. The maximum soluble concentration - at which no aggregates are observed - is indicated and the colour code indicates if the compound forms aggregates at the IC 50 concentration (green - no aggregation at IC 50 concentration; red - aggregation at IC 50 concentration.
  • the structure-activity relationship (SAR) scoping was directed at establishing the limitations of the tool chemotype, elucidating what kind of substituents were tolerated in each position and establishing the optimal vector and positioning of different functionalities.
  • the substituents to the cyclic group -A- play an unexpected relevant role for the activity, affording different reactivity patterns arising from non-covalent interactions.
  • Introducing a strong electron donating group like methoxy (OMe) (9E) as a substituent to a phenylene cyclic group decreases potency by 4-fold while the weak electron donating group methyl has different effects depending on its positioning about the phenylene ring.
  • the binding process is partially regulated by the nucleophilic attack at the acceptor group
  • the carbamate functionality (16) is related to amide-ester hybrids as it participates in hydrogen bonding through the carboxyl group and the backbone NH. Its ability to modulate inter- and intramolecular interactions prompted us to use this functionality, also reinforced by the chemical and proteolytic stability, and ability to permeate cell membranes (Ghosh et a!.).
  • 6-substituted analogues compounds 9B and 9B were chosen as appropriate comparators. These are the compounds with the phenylene cyclic group -A- is unsubstituted (9D) or is substituted with methyl at the 4-position (9B).
  • the core has a phenyl substituent at the 6-position, and that phenyl is itself substituted with a sulfonamide, which drastically reduces cLogP while increasing solubility and permeability.
  • Ligand efficiency (LE) and lipophilic efficiency (LipE) are two important metrics of “druglikeness” which are associated with improved prospects for good drug properties such as e.g. bioavailability. These depend on the molecule’s activity and physicochemical properties and are used as criteria for progression of the most promising candidates across drug discovery pipelines (Bembenek et ai ⁇ Perola; Hann et a!.).
  • LE is used to compare binding efficacy of inhibitors/ligands relative to their size while LipE is used as comparative binding efficacy taking into consideration the lipophilicity of the molecules.
  • BMX-IN-1 and analogues 24-26 only 27 reflects a major improvement in LipE, empowered by the drastic reduction in cLogP due to the introduction of an aliphatic amine.
  • the LE improvement is driven by the increased potency of all analogues rather than a decrease in the size of the molecule.
  • BMX inhibitors also display the ability to inhibit Bruton’s tyrosine kinase (BTK).
  • BTK tyrosine kinase
  • BMX-IN-1 displays 7-fold higher IC50 against BTK when compared to BMX. Consequently, the new analogues offer a greater improvement of LE and LipE metrics regarding BTK binding, in comparison to BMX-IN-1.
  • Table 2 Biochemical IC50, LE and LipE for compounds BMX-IN-1 and 24-27 against BMX and BTK.
  • BMX kinase activity was performed at CEREP-France. Briefly, the inhibition of the human recombinant Bmx kinase is quantified by measuring the phosphorylation of the substrate ⁇ o ⁇ I-bAbAbAEEEROUEEIRIUI_EI_I_R using a human recombinant enzyme expressed in insect cells and the HTRF detection method. The compounds for testing were incubated for 60 min at room temperature and the results expressed as a percent of control specific activity. BTK kinase activity (IC50) was performed at Discover-X through a radiometric assay.
  • DSF a fluorescence-based thermal shift assay
  • the purified recombinant human HiS 6 -BMX protein was subjected to thermal scanning in the absence and presence of the experimental compounds described herein, and the protein melting temperature (T m ) was calculated from the melting curve.
  • BMX-IN-1 increases the Tm value by 8.04°C.
  • Compounds 11 , 12 and 13 showed virtually no changes in the protein melting temperature, suggesting that a low affinity or no interaction at all may be occurring, corroborating the results obtained in the enzymatic assays.
  • Compounds 24 and 27 stabilized the protein increasing Tm by ⁇ 11.34°C and ⁇ 10.81°C, once more suggesting the direct binding of 24 and 27 to BMX with higher affinity.
  • DSF was performed in MicroAmpTM EnduraPlateTM Optical 96-Well Clear Reaction Plates with Barcode (Applied Biosystems, Life Technologies, California, USA) using a
  • SPR Surface Plasmon Resonance
  • His6-BMX protein was diluted to 10 pg/mL in sodium acetate pH 5.5, in the presence of 5 mM staurosporine and immobilized onto CM5 (Series S) sensor chips, using the standard amine coupling procedure. Prior to immobilization, the carboxymethylated surface of the chip was activated with
  • HBS-N 10 mM HEPES pH 7.4, 150 mM NaCI
  • Protein was coupled to the surface after 2 to 10 min injection times, at a flow rate of 10 pL/min, in order to reach 1 ,500 to 3,500 response units (RU). The remaining activated carboxymethylated groups were blocked with a 7 min injection of 1 M ethanolamine pH 8.5.
  • a protein sample was buffer exchanged to 200 mM ammonium acetate, pH7.6 and analyzed on a modified Q-exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) [69] using gold-coated glass needles (Hernandez et ai).
  • Typical native MS settings are a source fragmentation voltage of 50 V and capillary temperature of 30°C.
  • Denaturing MS analysis of drug conjugated protein was performed by liquid chromatography-MS (LC-MS) using a Dionex UltiMate 3000 RSLC Nano system coupled with a LTQ Orbitrap XL hybrid ion trap-Orbitrap spectrometer (Thermo Fisher Scientific).
  • the protein sample was directly loaded onto a C18 trap cartridge (Acclaim PepMap100, C18, 1 mm x 5 mm Thermo Scientific), desalted with 100% buffer A (100%
  • Typical MS conditions were a spray voltage of 1.8 kV and capillary temperature of 300°C.
  • the LTQ-Orbitrap XL was set up in positive ion mode with ion trap scanning (m/z 335-2000).
  • the proteomics analysis of drug conjugated protein was performed on the same LC-MS system with minor changes. Tryptic digested peptides were loaded to a C18 trap cartridge, desalted with 100% buffer A (100% H O and 0.1% formic acid) at 20 pl/min for 5 min and separated on a C18 analytical column with a linear gradient from 0% to 60% buffer B (80% acetonitrile, 20% H O and 0.1% formic acid) at flow rate of 300 nL/min.
  • LTQ-XL was operated in data-dependent acquisition mode with one full MS scan followed by 5 MS/MS scans with collision-induced dissociation.
  • full MS scan the mass range was set to 335 to 2,000 m/z at a resolution of 60,000.
  • tandem MS scan the CID normalized energy was 35%.
  • Crystals were grown through co-crystallization of BMX protein with the inhibitor 24 (details given below).
  • the X-ray crystal structure of BMX in complex with inhibitor 24 was determined to 2.0 A resolution, with a well-defined electron density map around the BMX ATP binding pocket, where the inhibitor is bound.
  • the values of the equivalent isotropic atomic displacement parameters for the ligand atoms within the pocket are comparable to those of the protein atoms they are interacting with, an indication of full ligand occupancy of the binding site.
  • an increase is observed in the sulfonamide aromatic ring, since this group is more exposed to the solvent and hence more mobile.
  • the crystal structure shows the expected covalent binding between the acrylamide warhead and Cys496 see (see Figure 3(A)).
  • Other major interactions of the inhibitor with the enzyme active site are mediated through polar non-bonding interactions between the nitrogen in the quinoline ring and Ne492 and quite unexpectedly between Lys445 and the oxygen located in the fused pyridinone ring (see Figure 3(B)).
  • the interaction with Lys445 is not observed at the sulfonamide group provided on the phenyl substituent to the core, but at the tricyclic quinoline core itself.
  • the polar interaction between 24 and Lys445 is actually one of the key points to regulate BMX activity.
  • the conserved b3 Lys interacts with aC-helix Glu residue in order to form a salt bridge required for ATP catalysis.
  • the binding of 24 to Lys445 alters this interaction between the b3 Lys and the aC-helix Glu and consequently inactivates BMX.
  • Other hydrophobic interactions occur between the aromatic rings of 24 and the side chains of Tyr491 , Ala443, Val431 , and Leu543 (data not shown).
  • Compound 24 is further stabilized by a hydrogen bond between a water molecule and the carbonyl oxygen of the acrylamide group.
  • a second water molecule stabilizes the first via a hydrogen bond, and forms hydrogen bonds with the peptide nitrogen of Cys496 and the terminal amine group of Asn499.
  • the crystal structure also shows that the DFG-motif adopts an out-like conformation
  • BMX DFG-motif is reminiscent of an inactive conformation or DFG-out, typically found in BTK and other kinases inactive structures (Sultan et a!), and it is also commonly observed in type II inhibitor complexes (Zhao et ai) [58] (data not shown).
  • the better-shaped crystals were analyzed at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France.
  • An X-ray diffraction data set to 2.0 A was collected at ESRF beamline ID30A-3 with a Dectris EIGER X 4M detector from a cryocooled crystal at 100 K.
  • the diffraction data were processed with AutoPROC and XDS (Ruegg et a!.).
  • Two diffraction datasets were obtained: in the first, a spherical region of reciprocal space to 2.2 A resolution was defined, and in the second a triaxial ellipsoidal region to a maximal resolution of 1.95 A was selected with the STARANISO module of AutoPROC (Ruegg et al.).
  • the structure of hBMX in complex with ligand 24 was determined by molecular replacement with PHASER (Vonrhein et at.) as implemented in the CCP4 program suite (McCoy et ai
  • stereochemical restraint dictionary for ligand 24 was created with JLIGAND (Murshudov et a/.) and the ligand was manually fitted into the electron density using COOT. Refinement was continued with PHENIX (Lebedev et at), alternating with manual model editing in COOT between refinements against sA-weighted 2
  • Each BMX molecule was divided into 4 rigid-body segments, estimated from the TLSMD server (Adams et at.) using the isotropic atomic displacement parameters from a previous refinement run. The final refinement was carried out to 2.0 A against the STARANISO dataset. Figures were prepared with PYMOL (Painter et a/.).
  • BMX belongs to a restricted group including other 10 kinases that share an equivalently placed cysteine in the ATP binding pocket.
  • This group comprises members from the TEC family (BTK, ITK, TXK and TEC), the EGFR family (EGFR, Her2, Her4), JAK3, BLK and dual specificity mitogen-activated protein kinase kinase 7 (MAP2K7). Therefore, the TEC, EGFR and JAK families were included in the screening, as well as the Src family and Lkb1 , which also have a cysteine within the same sequence alignment.
  • kinases involved in upstream Src, FAK, PI3K, mTOR, PDK1
  • Akt, PAK1 , TAM downstream kinases involved in upstream regulation of BMX signalling pathway and nonreceptor tyrosine protein kinase Abl.
  • the KinomeScan platform is a binding assay and the screening showed that compound 25 shows strong binding affinity against all the members of TEC family that share an equivalently placed cysteine and within these, higher affinity is observed towards BMX, BTK and TEC (see Table 5 below).
  • Table 5 Kinase Selectivity of Compound 25 using KinomeScan Technology.
  • the TEC family has high sequence similarity and in particular residues in the ATP binding kinase domain share 40-65% identity and 60-80% similarity.
  • the ATP binding sites are also highly conserved between the TEC and Src families with 14 identical residues out of 18 that comprise the ATP binding pocket.
  • BMX shares a 57% similarity to Src and most importantly, one of the key determinants of kinase selectivity - the gatekeeper residue - is a Thr in both the Src family and the TEC family members except ITK [56] It is therefore not surprising that 25 also binds Blk (and JAK3) while no affinity was observed with other targets.
  • BMX The role of BMX in different pathologies is not yet fully validated. Notwithstanding, it has been implicated in many regulatory mechanisms and despite the absence of a BMX dependent disease model, prostate cancer cell lines have been used to evaluate inhibitors anti-proliferative effects in a cellular context. The activation of BMX in response to PI3K signalling is just one of the mechanisms through which the levels of BMX became increased in prostate cancer (Chau et ai ⁇ Guo et at).
  • the androgen-receptor negative PC-3 cells are resistant to the treatment, with no significant anti-proliferative effect at the maximum concentration tested (10 mM).
  • BMX-IN-1 and 24 showed a GI50 of 1.4 pM and 2.8 pM, respectively.
  • Compound 27 was the least active (GI50 10 pM) while 25 and 26 showed a GI50 around 5 pM, as shown in Table 6.
  • Table 6 Antiproliferative activity of compounds BMX-IN-1 and 24-27 against LNCaP and PC-3 prostate cancer cell lines.
  • LNCaP and PC-3 Proliferation in LNCaP and PC-3 was measured following 96 h incubation with the drugs. GI50 values are reported in pM and are the mean of three individual experiments performed in triplicate. Cells were seeded in white, opaque-bottom 96-well plates at 5,000 cells/well (LNCaP) or 2000 cells/well (PC-3) in a total volume of 100 pl_ of culture media. Serial diluted
  • BMX-IN-1 against RV-1 cells could only be potentiated with the Akt inhibitor MK2206 (Liu et a! ⁇ another inhibitor, ABT-737 only induces apoptosis upon co-treatment with PI3K inhibitors (Li et ai) ⁇ the dual BMX/BTK inhibitor CTN06 requires a co-treatment with the autophagy inhibitor chloroquine (CQ) or docetaxel to inhibit PC-3 cells growth (Guo et at.) and a similar profile is observed with the dual BMX/Src inhibitor CTA095 synergizing with CQ and paclitaxel (Guo et at).
  • CQ chloroquine
  • CTA095 synergizing with CQ and paclitaxel
  • LNCaP cells were seeded in 24 well-plates at 8,000 cells/well in in a total volume of 500 pL of culture media and incubated for 24 hours to allow for attachment. After this time, 5 mM of each compound diluted in culture medium was added to the cells. After 64 hours of treatment, cells were harvested after trypsinization (TrypLE Express, LifeTechnologies,
  • the inactivation of BMX occurs in a two-step process that is governed by two parameters: the affinity of the initial non-covalent binding, Kl, and the rate of the subsequent covalent bond-forming reaction with the thiol of the cysteine residue, ki nact .
  • the rate of inactivation (kinact/KI) is second-order, which describes the efficiency of covalent bond formation.
  • the enhanced activity must be a result of the structural modifications introduced in the scaffold.
  • Table 8 Intracellular target engagement in HEK293 cells transiently transfected with BMX expressing NanoLuc ® -BMX.
  • HEK296 cells purchased from ATCC, were transfected with BMX and treated in duplicate with test compounds, BMX-IN-1 or JS25, and with the reference compound Dasatinib, for 1 hour of incubation. Compounds were diluted 10 times with 3-fold dilution, starting at 1 mM. Curve fits were performed only when % NanoBret signal at the highest concentration of compounds was less than 55%. The IC50 values were determined using the GraphPad Prism 8 (USA).
  • BMX-IN-1 The role of BMX in different pathologies is not yet fully validated. Nevertheless, it has been implicated in many regulatory mechanisms and despite the absence of a BMX dependent disease model, prostate cancer cell lines have been used to evaluate anti-proliferative effects of the inhibitors in a cellular context.
  • a previous experiment we screened several inhibitors in a collection of cell lines representing prostate, brain, blood, breast, ovary, lung, bone marrow and lymphoid tumour tissues. Compounds were incubated with cells for 72 h in a 386 well-plate format to monitor dose-dependent impact on viable cell growth by using the CellTiter-Glo ® luminescent assay, which quantifies ATP and the presence of metabolically active cells.
  • BMX-IN-1 demonstrated more potent inhibitory effects relative to 24 in the four prostate cancer cell lines that were included in the panel, 22RV1 , PC3, LNCaP and DU145, particularly in those dependent on androgen receptor signaling (LNCaP and 22RV1).
  • LNCaP and 22RV1 dependent on androgen receptor signaling
  • DU145 and PC3 were overall more resistant to treatment.
  • 24 showed potent inhibitory effects against LNCaP and 22RV1 but also against PC3, which are androgen receptor negative cells.
  • Table 9 Viable cell growth inhibition of compounds BMX-IN-1 , 10, 11 and 24 in a panel of prostate, brain, blood, breast, ovary, lung and lymphoid cancer cells.
  • Compound activity was profiled against 14 human cell lines from different tissues in a 384- well format, opaque white assay plates at 500-1000 cells per well using a semi-automated system. Cells were incubated at 37°C and 5% CO2. Compound stocks were plated in a 384- well format in 11-point and 2-fold concentration ranges. Compounds were pin-transferred into duplicate assay plates and incubated for72h. ATP levels were assessed by CellTiter- Glo ® (Promega) according to the manufacturer’s instructions. The values were normalized to vehicle and GI50 was calculated using GraphPad Prism 8. When ambiguous fit was observed curves were top (100%) and bottom (0%) constrained and GI50 was determined with 4-P least squares fit. In these cases SD is not calculated by GraphPad Prism 8.
  • BMX inhibition alone induces limited cell death in BMX-expressed cell lines owing to the existence of compensatory mechanisms in signalling pathways.
  • BMX inhibitors in combination treatment regimens, the synergistic anti-proliferative effects of BMX inhibitors when combined with other therapeutic agents, which pre-sensitize prostate cancer cells was examined.
  • LNCaP cells were co-treated in a combinatorial fashion with compounds 24-26, AKT1/2 (AKT inhibitor), Flutamide (androgen receptor antagonist) and LY294002 (PI3K inhibitor). Cell viability was evaluated after 5 days with CellTiter-Glo ® and compared with the overall anti-proliferative effects of the compounds alone.
  • LNCaP cells were seeded in 96 well-plates at 5000 cells/well in in a total volume of 100 pL of culture media and incubated for 24 hours to allow for attachment. After incubation, cells were treated in triplicate, in a combinatorial-fashion with 24 (2 pM and 3 pM), 25 (5 pM and 6 pM), 26 (6 pM), AKT1/2 (1 pM and 2 pM), Flutamide (25 pM and 50 pM), and PI3K inhibitor (3 pM and 3.5 pM). The results are shown in Figure 5.
  • Compound 25 was tested through 1 1 Diffuse Large B-cell lymphoma (DLBCL) samples from hospital patients to quantify its ability to induce targeted cell cytotoxicity on the B-cancer cell fraction versus non-transformed cells.
  • CD20+CD79a+ markers double and single positive cells were used to determine the target cancer fraction. Results are shown in Figure 6.
  • Figure 6A shows the relative cell fraction (RCF) of the viable target cells for increasing concentrations of 25 in DMSO. Relative cell fraction of ⁇ 1.0 (hashed line) indicates on-target cytotoxic response, >1.0 indicates general cytotoxicity or off-target cytotoxic response.
  • Figure 6B shows this normalized to the fraction of target cell population at increasing DMSO concentrations.
  • Figure 6C shows 11 primary patient samples ranked by the drug response score (DRS) of compound 25 calculated as 1-mean of the RCF.
  • DRS drug response score
  • Data is 1 1 biological repeats, each concentration point for each sample was performed in 4 replicates, at a single 72 h hour incubation time point.
  • the drug response score (DRS) has been previously shown to correlate to clinical response for late stage hematological cancer patients (Snijder et ai).
  • Viable target cells are defined as cytotoxicity-marker negative and diagnostic marker (CD19, CD20, and/or CD79a) positive B-cells.
  • compound 25 has an“on target” effect in 7 out of 1 1 patient samples.

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