WO2020245430A1

WO2020245430A1 - Benzo[h][1,6] naphthyridin-2(1h)-ones as bmx inhibitors, for use against cancer

Info

Publication number: WO2020245430A1
Application number: PCT/EP2020/065730
Authority: WO
Inventors: João SEIXAS; Gonçalo BERNARDES
Original assignee: Instituto De Medicina Molecular Joao Lobo Antunes
Priority date: 2019-06-07
Filing date: 2020-06-05
Publication date: 2020-12-10
Also published as: CN114206867A; AU2020288706A1; CA3140767A1; GB201908171D0; EP3980126A1

Abstract

The present invention provides a compound of formula (I) and its use in methods of treatment, including the treatment of cancer. The compound has the structure shown below where D is an acceptor group and -A-, -L-, R⁷ are as discussed in the application: Formula (1a). The present invention also provides a compound of formula (II) and its use in methods of treatment including the treatment of cancer. The compound has the structure shown below where D is an acceptor group and -A-, -L-, R6 are as discussed in the application Formula (1b).

Description

BENZO[H][1 ,6] NAPHTHYRIDIN-2(1 H)-ONES AS BMX INHIBITORS, FOR USE AGAINST

CANCER

Related Application

The present case claims the benefit of, and priority to, GB01908171.0 filed on 7 June 2019 (07.06.2019), the contents of which are hereby incorporated by reference in their entirety.

Field of the Invention

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.

Also provided are complexes comprising a compound of the invention covalently bound to a polypeptide, such as covalently bound to BMX, and polymorphs of these complexes.

Background

Over the last couple of years, the development of irreversible kinase inhibitors has gained more traction both in academia and the pharmaceutical industry (Chaikuad et al:, Singh et al. 2018). Historically, irreversible inhibitors were considered problematic due to their lack of selectivity and safety issues related to the undesired engagement of off-targets. However, these potential liabilities can be overcome, and the development of covalent small molecule kinase inhibitors has recently seen renewed interest (Singh et al:, Barf et al. ] Bourne et al:, Lagoutte et al .; Gilbert et al.). Supporting the value and“renaissance” of covalent inhibitors, four small molecule entities have recently been approved by the FDA for clinical use: Afatinib (EGFR and HER2 inhibitor), Ibrutinib and Acalabrutinib (BTK inhibitor), Osimertinib (EGFR inhibitor) and Neratinib (EGFR and HER2 inhibitor) (Byrd et al.; Honigberg et a /.; Rabindran et al.; Soria et al:, Miller et al.). However, not all kinases are accessible for covalent binding, as it depends on the target amino acid positioning (Zhang et al:, Liu et al. Chem. Biol. 2013; Zhao et al:, Lanning et al.). One of such kinases of interest is the epithelial and endothelial tyrosine kinase (ETK), commonly known as bone marrow tyrosine kinase in chromosome X (BMX).

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. Moreover, 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.).

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

Wen et al:, Guryanova et al:, Kaukonen et al:, Mano et al.). Moreover, it has been found to be overexpressed in numerous cancer types including breast (Bagheri-Yarmand et al:,

Chen et a!:, Cohen et al.), prostate (Dai et al. Cancer Res. 2006; Dai et al. Cancer Res. 2010), colon (Potter et al. Neoplasia 2014) and cervical carcinoma (Li et al.), suggesting that elevated levels of BMX increase cancer cell survival. In addition, 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.).

Therefore, taking into consideration the existence of multiple downstream target proteins, the integration in multiple and diverse signalling pathways, together with the fact that it regulates proliferation, migration and antiapoptotic effect, BMX emerges as a potential target for multiple aspects of cancer therapy. Recent studies also highlighted that modulation of BMX activity sensitizes cells to therapeutic agents improving response to chemotherapy DNA damaging agents or radiation. These studies show strong evidence that both direct inhibition of BMX or through modulation of related pathways result in an increased therapeutic efficacy (Potter et al. Neoplasia 2014; Fox et al:, Potter et al. Mol. Cancer Ther. 2016).

Several Endothelial Growth Factor Receptor (EGFR) inhibitors in clinical development were shown to irreversibly alkylate BMX at a unique cysteine residue in the aforementioned fashion (Hur et al.). These molecules are derived from reversible EGFR receptors gefitinib (Iressa®) and erlotinib (Tarceva®), by adding a Michael acceptor moiety that reacts with the cysteine residue (Cys496) in the ATP binding site. This cysteine residue is a unique occurrence found in the ATP binding pocket and is present in all five members of the TEC-family kinase members. Therefore, by virtue of structural homology these compounds could also be covalent inhibitors of the other kinases in the TEC family.

BMX-IN-1 is one of the most potent BMX inhibitors (IC₅₀: 8.0 nM) reported in the literature and also binds to BTK with very high affinity (IC₅₀: 10.4 nM) (Liu et al. ACS Chem. Biol.

2013).

WO 2014/063054 describes compounds for use as inhibitors of Bone Marrow Tyrosine Kinase on Chromosome on X (BMX). 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. ( Scientific Reports) 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.

In further work, Wu et al. ( ACS Chem. Biol.) also show that the same compound is an inhibitor for B-Cell lymphoma.

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

WO 2014/063054 and Liu et al. 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.

Certain compounds are shown to have antiproliferative activity against a panel of cancer cell lines, including lung cancer, prostate cancer and colorectal carcinoma cell lines. Jarboe et al. and Liang et al. are reviews of the known inhibitors of BMX and BTX

respectively. The inhibitors disclosed in these reviews differ from those described in the literature cited above.

There is a need for alternative compounds for use in treating cancer, for example where such compounds covalently bind kinases such as BMX, BTK and TEC.

Summary of the Invention

In a general aspect 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.

Also provided are related compounds where the 6-position of the quinoline ring is

substituted, rather than the 7-position. 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.

Accordingly, in a first aspect of the invention there is provided a compound of formula (I):

and salts, solvates and protected forms thereof, wherein:

-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; and -R⁷ 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)₂NH-, *-S(0)₂N(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,

C2-6 alkenylene, C2-6 alkynylene and C2-6 heteroalkylene; and

-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.

In a second aspect of the invention there is provided a compound of formula (II):

and salts, solvates and protected forms thereof, wherein:

-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; and

-R⁶ is -|_^6A-|_^6B-R^6A, where -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)₂NH-, ^*-S(0)₂N(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,

C_2-6 alkynylene and C_2-6 heteroalkylene; and

-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.

Optionally, the compounds of formula (II) are not either of the following compounds:

In a third aspect of the invention, there is provided 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.

In a further aspect 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.

In yet a further aspect 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.

In a further aspect 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.

In one aspect 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.

In a further aspect there is provided a method of forming a complex, the method 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.

In each of these aspects the polypeptide may be a protein, such as a kinase, such as BMX or BTK.

These and other aspects and embodiments of the invention are described in further detail herein.

Summary of the Figures

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). A) Cells co-treated with 24 (3 mM), 25 (5 mM) and 26 (6 pM) with AKT1/2 (1 pM); B) 24 (3 pM), 25 (5 pM) and 26 (6 pM) with Flutamide (50 pM); C) 24

(3 pM), 25 (5 pM) and 26 (6 pM) with LY294002 (3 pM). Values are reported in % cell viability normalized to DMSO controls and are the mean of three individual experiments performed in triplicate. Determined P-values are illustrated as ns (P>0.05), * (P £ 0.05), **

(P £ 0.01), *** (P £ 0.001) and **** (P £ 0.0001).

Figure 6 shows the induced targeted cell cytotoxicity on the B-cancer cell in primary DLBCL samples. A) Relative cell fraction (RCF) of the viable target cells for increasing

concentrations of 25 in DMSO; relative cell fraction is the percentage of the target cell population. B) Normalized to the fraction of target cell population at increasing concentration in DMSO. C) 11 primary patient samples ranked by the drug response score (DRS) of compound 25 calculated as 1-mean of the RCF.

Detailed Description of the Invention

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 compounds of formula (I) and (II) are described in further detail below.

Compounds of Formula (!)

The invention provides a compound of formula (I):

and salts, solvates and protected forms thereof, wherein:

-L- is a covalent bond or Ci-e alkylene;

-D is an acceptor group, such as a Michael acceptor group; and

-R⁷ 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)₂NH-, ^*-S(0)₂N(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,

C2-6 alkynylene and C2-6 heteroalkylene; and

-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.

Compounds of Formula (II)

The invention also provides a compound of formula (I):

and salts, solvates and protected forms thereof, wherein:

-L- is a covalent bond or Ci-e alkylene;

-D is an acceptor group, such as a Michael acceptor group; and

-R⁶ is -L^6A-L^6B-R^6A, where

-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)₂NH-, ^*-S(0)₂N(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,

C_2-6 alkynylene and C_2-6 heteroalkylene; and

The substituent groups and the optional substituents groups for -R^6A are described in further detail below. In one embodiment, the compound of formula (II) may not be:

In one embodiment, the compound of formula (II) may not be:

The compounds identified above are disclosed in Liang et al. as compounds 7 and 8 (Liang et al. 2017). Compounds from this document are also discussed in WO2013154778. These compounds are tested for their antiviral activity against the dengue virus in a viral focus-forming assay and a viral protein accumulation assay. They are not disclosed as suitable for use in the treatment of any disease associated with altered kinase activity, such as proliferative diseases, and they are not said to bind TEC, or any other kinase.

This exclusion applies only to those aspects of the present invention that relate to compounds of formula (II), compositions containing compounds of formula (II) and the uses of such compounds and compositions in methods of medical treatment, particularly insofar as they relate to the use in antiviral treatments, such as treatment of a dengue virus infection.

-A-

The group -A- is a cyclic group which is a substituent at the nitrogen ring atom of the pyridinone ring within the tricyclic core.

The worked examples in the present exemplify the use of phenylene as the cyclic group -A-. It is known from the art that other cyclic groups may be used at this position. For example, 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). Thus, 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. Preferably 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.

Where -A- comprises two or more fused rings, it is preferred that 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.

Where -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).

Where a number of atoms is given, this refers to the total number of ring atoms, including carbon and hetero (nitrogen) ring atoms as appropriate.

Where -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. A

cycloalkylene group may be partially unsaturated (but not aromatic).

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 cycloalkylene group may be selected from cyclopentylene (C₅), cyclohexylene (Ob);

tetralinylene and decalinylene (C₁₀), such as cyclohexylene.

Where -A- is heterocyclylene this may be C3-10 heterocyclylene, such as

C5-10 heterocyclylene. 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,

tetrahydrothiophenylene, pyrrolinylene (C5) ; piperidinylene, piperazinylene,

tetrahydropyranylene, dioxanylene, thianylene, dithianylene, morpholinylene and thiomorpholinylene (Ob); indolinylene, decahydroisoquinolinylene, decahydroquinolinylene and tetrahydroquinoline, and tetrahydroisoquinoline (C10).

Where the cyclic group has 6 ring atoms only, 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).

Where the cyclic group has 6 ring atoms only, 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). Preferably the cyclic group is not substituted at the 2- or 6-positions. Typically, 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.

Preferably, the group -A- is an optionally substituted group selected from phenylene, pyridinylene, indolylene, isoindolylene, benzoimidazolylene, indolinylene, isoindolinylene, tetrahydroquinolinylene and tetrahydroisoquinolinylene.

More preferably, 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. Where -A- is phenylene, this may be phenyl-1 , 3-ene. Here, the 1-position is the carbon ring atom attached to the pyridone nitrogen.

Where the 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. Preferably, the phenylene is monosubstituted, and is substituted at the 4-position.

When -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. Here, the 1-position is the nitrogen ring atom.

Preferably, 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.

When -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. Here, 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.

Where -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. Here, 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.

Where -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. Here, the benzoimidazolylene may be connected to pyridine via the 6-position.

The benzoimidazolylene is unsubstituted or monosubstituted with -R^A. The

benzoimidazolylene is preferably substituted on the benzene ring with -R^A.

When -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. Here, 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.

Where -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. Here, 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. Where -A- is tetrahydroquinolinylene (or 1 ,2,3,4-tetrahydroquinolinylene) this may be tetrahydroquinolinyl-1 ,7-ene, where the 1-position is nitrogen ring atom. The

tetrahydroquinolinylene may be connected to -L- via the nitrogen ring atom. Here, the tetrahydroquinolinylene may be connected to pyridine via the 7-position. The

tetrahydroquinolinylene is unsubstituted or monosubstituted with -R^A. The

tetrahydroquinolinylene is preferably substituted on the benzene ring with -R^A.

Where -A- is tetrahydroisoquinolinylene (or 1 ,2, 3, 4- tetrahydroisoquinolinylene) this may be tetrahydroisoquinolinyl-2,6-ene, where the 1-position is nitrogen ring atom. The

tetrahydroisoquinolinylene may be connected to -L- via the nitrogen ring atom. Here, the tetrahydroisoquinolinylene may be connected to pyridine via the 6-position. The

tetrahydroisoquinolinylene is unsubstituted or monosubstituted with -R^A. The

tetrahydroisoquinolinylene is preferably substituted on the benzene ring with -R^A.

Preferably, the group -A- is optionally substituted phenylene, such as phenylene substituted with one further substituent -R^A. Most preferably the phenylene is phenyl-1 , 3-ene, optionally substituted at the 4-position. Here, 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.

-R^A

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. In one embodiment, the cyclic group -A- is not substituted with -R^A, it is monosubstituted with -R^A or it is disubstituted with -R^A. Preferably, however, the cyclic group is not substituted with -R^A, or it is monosubstituted with -R^A.

In the worked examples of the present case, 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.

Where -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 (-NH₂), 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.

Where -R^AA is optionally substituted, each optional substituent may be selected from halo, hydroxy (-OH), amino (-NH₂), 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. Thus, here 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 C₂-e alkynyl, such as ethynyl or propynyl.

An 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.

Preferably, 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.

-L-

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.

In the worked examples of the present case the group -L- is a covalent bond.

It is known from the art that other linkers may be used to connect the acceptor group to the cyclic group. For example, 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). Thus, the compounds of the present case are not limited to those where the cyclic group -A- connects directly to -D.

Where -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.

Preferably -L- is a covalent bond. Here, the cyclic group -A- connects directly to -D.

-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.

In the present case, 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. Here, the compound is covalently linked to the kinase via the acceptor group and a site on the kinase.

In the worked examples of the present case, 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. For example, WO 2014/063054 describes compounds having a range of acceptor groups, (these are the groups -R^D and -R^G in this prior art). Thus, 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-).

When the group -A- is connected to -L^M- via a nitrogen ring atom (for example, when -L- is a covalent bond), -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. Typically, the reactive functionality is electrophilic, and more specifically, together with group -X-, it is a Michael acceptor group.

Where -M is alkenyl this may be C_2-6 alkenyl. The alkenyl group may contain one carbon- carbon double bond. Preferably 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).

Where -M is alkynyl this may be C_2-6 alkynyl. The alkynyl group may contain one carbon- carbon triple bond. Preferably the triple bond is conjugated with a carbonyl group present as -X-.

Where -M is 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. Preferably, where the carbonyl is present, 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. Where the heterocyclyl is a C3 heterocyclyl it is preferably unsaturated.

A C₃ heterocyclyl may be selected from aziridinyl, oxiranyl and thiiranyl.

Where the heterocyclyl is a C₅ heterocyclyl it is preferably partially saturated, and preferably contains a single carbon-carbon double bond.

In one embodiment -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.

In one embodiment, -M is selected from optionally substituted alkenyl, optionally substituted alkynyl, cyano, and alkyl substituted with cyano.

It is preferred that -M is optionally substituted alkenyl, more preferably optionally substituted ethenyl, and most preferably ethenyl (-CH=CH2).

The group -D, such as -L-D together, may be selected from -N(H)C(0)CHCH₂,

-N(H)C(0)CH₂CN, -N(H)C(0)CCH, -N(H)C(0)CN, -N(H)C(0)CHCHMe, and

-N(H)C(0)CCMe.

The group -D, such as -L-D together, is preferably -N(H)C(0)CHCH₂.

For the avoidance of doubt, the group -CF₃ is not an acceptor group. Thus, -D cannot be -CF₃.

-R⁷

The group -R⁷ is a substituent at the 7-position of the quinoline ring. In contrast, 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.

The inventors understand that those substituents previously used at the 6-position may be provided alternatively at the 7-position. Thus, 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. Thus, -R⁷ may contain aryl. Here, 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.

Moreover, alternative groups may be used at the 7-position, where such groups have never been described for use at the 6-position. As noted above, 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 -R⁷ 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)₂NH-, *-S(0)₂N(R^N)-, *-NHS(0)₂- and *-N(R^N)S(0)₂-, where -R^N is C1-6 alkyl and the asterisk indicates the point of attachment to the quinoline;

-L^7B- is a covalent bond or selected from C1-6 alkylene, C_2-6 alkenylene,

C_2-6 alkynylene and C_2-6 heteroalkylene; and

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.

Preferably, the group -R⁷ contains a nitrogen atom. Such may be provided where -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. Alternatively, the group -R^7A may be connected directly to the quinoline ring. In this case, each of -L^7A- and -L^7B- is a covalent bond.

Preferably, each of -L^7A- and -L^7B- is a covalent bond. Here -R⁷ is -R^7A. Thus, -R^7A is connected directly to the tricyclic ring.

In another embodiment, -R⁷ is -L^7B-R^7A, and preferably -L^7B- is C_2-6 alkenylene.

In another embodiment, -R⁷ is -L^7A-R^7A, and preferably -L^7A- is -NH- or -N(R^N)-. When -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.

In one embodiment, -R^7A is optionally substituted aryl, such as optionally substituted phenyl, pyridinyl, pyrrolyl, oxazolyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, quinolinyl and isoquinolinyl.

Preferably, -R^7A is optionally substituted phenyl or pyridinyl, such as phenyl. The phenyl or the pyridinyl is optionally substituted, such as optionally monosubstituted.

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.

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).

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.

A cycloalkyl group may be selected from cyclopentylene (Cs), cyclohexylene (Ce);

tetralinylene and decalinylene (C₁₀), such as cyclohexylene. When -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.

When -R^7A is alkenyl it may be C_2-6 alkenyl, such as C₂-4 alkenyl, such ethenyl. The alkenyl group may be linear or branched. The alkenyl may be optionally substituted.

When -R^7A is alkynyl it may be C_2-6 alkynyl, such as C₂-4 alkynyl, such ethynyl. The alkynyl group may be linear or branched. The alkynyl may be optionally substituted.

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.

-R⁶

The group -R⁶ 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.

As noted above, two compounds having a non-aromatic group bound directly to the quinoline at 6-postion are known from Liang et al. (Liang et al. 2017). These compounds may be excluded from the definition for the compounds of formula (II).

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 -R⁶ is -|_^6A-L^6B-R^6A, where

-L^6B- is selected from a covalent bond or selected from Ci-e alkylene,

C_2-6 alkenylene, C_2-6 alkynylene and C_2-6 heteroalkylene; and -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 group -R⁶ 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 -R⁶ may not be optionally substituted piperazinyl, such as optionally substituted piperazin-1-yl. More specifically, -R⁶ 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.

Preferably, the group -R⁶ contains a nitrogen atom. Such may be provided where -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. Alternatively, the group -R^6A may be connected directly to the quinoline ring. In this case, each of -L^6A- and -L^6B- is a covalent bond.

Preferably, each of -L^6A- and -L^6B- is a covalent bond. Here -R⁶ is -R^6A. Thus, -R^6A is connected directly to the tricyclic ring.

In another embodiment, -R⁶ is -L^6B-R^6A, and preferably -L^6B- is C alkenylene.

In another embodiment, -R⁶ is -L^6A-R^6A, and preferably -L^6A- is -NH- or -N(R^N)-.

When -R^6A is heterocyclyl it may be C3-14 heterocyclyl, such as C5-7 heterocyclyl, such as C5-6 heterocyclyl.

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.

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).

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.

A cycloalkyl group may be selected from cyclopentylene (C₅), cyclohexylene (Ob);

tetralinylene and decalinylene (C₁₀), such as cyclohexylene.

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.

When -R^6A is 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.

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.

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.

In one embodiment, the group -R⁶ contains no aromatic functional group, for example, the group -R⁶ does not contain a phenyl group.

-R^s

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. Here, 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. Here, the group -R^s is -R^SN.

In one embodiment, 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.

In one embodiment, -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-.

In one embodiment, 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; and

-R^ss is selected from optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl and aryl.

In one embodiment, -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-.

Where -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.

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.

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.

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).

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.

tetralinylene and decalinylene (C₁₀), such as cyclohexylene.

When -R^ss 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^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.

When -R^ss is aryl it may be carboaryl or 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.

In one embodiment, -R^ss is optionally substituted aryl, such as optionally substituted phenyl, pyridinyl, pyrrolyl, oxazolyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, quinolinyl and isoquinolinyl.

Preferably -R^ss is alkyl.

Where -R^ss is optionally substituted, each optional substituent may be selected from the group consisting of halo (such as -F, -Cl, -Br, and -I), hydroxy (-OH), amino (-NH₂), 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. Exemplary Compounds of Formula (!)

The compounds of formula (I) may be a compound as set out below:

and salts, solvates and protected forms thereof, wherein -R^A, -R⁷ and -M have the same meanings as given above. Here, 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:

and salts, solvates and protected forms thereof, wherein -R⁷ and -M have the same meanings as given above.

The compounds of formula (I) may be a compound as set out below:

and salts, solvates and protected forms thereof, wherein -R⁷ and -M have the same meanings as given above. Exemplary Compounds of Formula (II)

The compounds of formula (II) may be a compound as set out below:

and salts, solvates and protected forms thereof, wherein -R^A, -R⁶ and -M have the same meanings as given above. Here, 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:

and salts, solvates and protected forms thereof, wherein -R⁶ and -M have the same meanings as given above.

The compounds of formula (II) may be a compound as set out below:

and salts, solvates and protected forms thereof, wherein -R⁶ and -M have the same meanings as given above. Complex

In one aspect, 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.

In one embodiment, 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, PIK3Cy and 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.

Preferably 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.

In one embodiment, 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.

Where the kinase is not a BMX kinase, the compound may be bound to a cysteine residue that corresponds to the Cys 496 residue of BMX.

Kinase - General Information

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. For example EMBOSS Needle pairwise sequence alignment can be used to determine amino acid sequence identity. EMBOSS Needle pairwise sequence alignment, which uses the Needleman-Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)), can be used to determine amino acid sequence similarity, for example using default parameters and using a BLOSUM scoring matrix such as the BLOSUM62 scoring matrix. Default parameters may be used with a gap creation penalty = 12 and gap extension penalty = 4. 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), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195- 197), generally employing default parameters.

Percent (%) amino acid sequence identity with respect to a reference sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent identity values may be determined by WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span = 1 , overlap fraction = 0.125, word threshold (T) = 11. 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

comparison to the number of amino acid residues in the reference sequence, after aligning the sequences.

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.

Alternatively or additionally, 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. Alternatively or additionally, 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, Solvates and Other Forms

Examples of 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.

In one embodiment 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. Examples of 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. In one embodiment the isotope is a stable isotope. Thus a compound described here includes, for example deuterium containing compounds and the like. For example, H may be in any isotopic form, including ¹H, ²H (D), and ³H (T); C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including ¹⁶0 and ¹⁸0; 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“isomeric forms”).

Note that, except as discussed below for tautomeric forms, specifically excluded from the term“isomers,” as used herein, 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). For example, a reference to a methoxy group, -OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta- chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., Ci-₆alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para- methoxyphenyl).

Unless otherwise specified, 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) and separation (e.g., fractional crystallisation and

chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

One aspect of the present invention pertains to compounds in substantially purified form and/or in a form substantially free from contaminants.

In one embodiment, 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.

Unless specified, the substantially purified form refers to the compound in any

stereoisomeric or enantiomeric form. For example, in one embodiment, the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds. In one embodiment, the substantially purified form refers to one stereoisomer, e.g., optically pure stereoisomer. In one embodiment, the substantially purified form refers to a mixture of enantiomers. In one embodiment, the substantially purified form refers to an equimolar mixture of enantiomers (i.e. , a racemic mixture, a racemate). In one embodiment, the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer.

In one embodiment, 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.

Unless specified, the contaminants refer to other compounds, that is, other than

stereoisomers or enantiomers. In one embodiment, the contaminants refer to other compounds and other stereoisomers. In one embodiment, the contaminants refer to other compounds and the other enantiomer.

In one embodiment, 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.

Methods of Treatment

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. In some aspects of the invention, 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. In one embodiment, 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

The term“treatment,” as used herein in the context of treating a condition, 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) is also included.

For example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term“treatment.”

The term“therapeutically-effective amount,” as used herein, pertains 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.

The term“treatment” includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.

Formulations

In one aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or (II) together with a pharmaceutically acceptable carrier.

While it is possible for the compound of formula (I) or (II) to be administered alone or together with the second agent, it is preferable to present it as a pharmaceutical formulation (e.g., composition, 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,

pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents. Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making 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.

The term“pharmaceutically acceptable,” as used herein, pertains to compounds,

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.

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.

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.

The compound may be dissolved in, suspended in, or admixed with one or more other pharmaceutically acceptable ingredients. Formulations suitable for oral administration (e.g., by ingestion) 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.

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.

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. Preferably, 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. Together, the

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. As an alternative method of administration, 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 (e.g., by inhalation or insufflation therapy) 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. Additionally or alternatively, a formulaton for pulmonary administration may be formulated for administration from a nebuliser or a dry powder inhaler. For example, 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.

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 (e.g., by injection), 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. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.

Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the 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

One aspect of the invention pertains to a kit 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

(b) 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.

In one embodiment, the kit further comprises (c) a second active agent, or a composition comprising the second active agent. Here, 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.

Routes of Administration

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

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., gorilla, chimpanzee, orang-utan, gibbon), or a human. Furthermore, the subject/patient may be any of its forms of development, for example, a foetus.

In one preferred embodiment, the subject/patient is a human.

It is also envisaged that the invention may be practised on a non-human animal having a microbial infection. 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.

Cell Treatment

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.

Examples of 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.

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.

Combination Therapy and Co-Treatment

A compound of formula (I) may be administered in conjunction with a second agent.

Administration may be simultaneous, separate or sequential.

The appropriate route of administration and how the compound of formula (I) and the second agent are administered will depend on the pharmacokinetics of the compound of formula (I) and the second agent.

By“simultaneous” administration, it is meant that a compound of formula (I) and a second agent are administered to a subject in a single dose by the same route of administration.

By“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.

By“sequential” it is meant that the two agents are administered at different points in time, provided that the activity of the first administered agent is present and ongoing in the subject at the time the second agent is administered.

Generally, 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. Alternatively, the active agent may be administered first, followed by the compound of formula (I).

Ultimately, 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. For example 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.

Other Preferences

Each and every compatible combination of the embodiments described above is explicitly disclosed herein, as if each and every combination was individually and explicitly recited.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example“A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.

Experimental and Results

General Methods and Materials

Starting materials were obtained from commercial suppliers and used without further purification unless otherwise stated. BMX-IN-1 was acquired from Calbiochem. Column chromatography was carried out with Merck Si60 (60-200 pm) silica gel columns as the stationary phase and analytical grade solvents as the eluent unless otherwise stated. All reactions using anhydrous conditions were performed under an argon atmosphere in oven- dried glassware.

Reactions were followed by thin layer chromatography (TLC) using coated silica gel plates (Merck, aluminium sheets, silica gel 60 coated with fluorescent indicator F254) and visualized by UV light and ninhydrin staining (if required). Proton magnetic resonance (¹H NMR) spectra were recorded at 300 MHz on a Bruker Fourier 300 spectrometer and are reported as follows: chemical shifts d (ppm) (multiplicity, coupling constant in J (Hz), number of protons). Multiplicities are labelled s, singlet; d, doublet; t, triplet; m, multiplet; br, broad; or a combination of these. Total ion current traces were obtained for electrospray positive and negative ionization (ES+/ES-) on a Waters Acquity QDa detector. Analytical chromatographic conditions used for the LC/MS analysis were as follows. The column was a Codecs C18 2.7 mM (4.6 mm x 50 mm). Solvent A was an aqueous solvent consisting of MilliQ water with 0.01 % Formic Acid and solvent B was acetonitrile with 0.01% Formic Acid. Additional chromatographic parameters were as follows: flow rate, 0.5 mL/min; injection volume, 5 pl_; column temperature, 40°C; and UV wavelength range, 210-400 nm. The purity of all tested compounds was ³95% using the analytical method described above unless otherwise stated.

High resolution mass spectrometry (HRMS) analysis were performed at the Unidad de Espectrometria de Masas e Proteomica of the Universidad de Santiago de Compostela. Samples were recorded on a Bruker Daltonics microTOF ESI-TOF mass spectrometer. Calculated and exact m/z values are indicated in Daltons.

Synthesis

Compounds

The compounds prepared in this study, including compounds of the invention and comparator compounds are set out in Table 1 below.

A compound of the invention includes compounds 24-27, where a group -R⁷ is present. These are compounds of formula (I).

A compound of the invention includes compounds 20-23, where a group -R⁶ is present. These are compounds of formula (II).

Compounds 9A-9E and 10-19 are provided as comparative examples for the useful understanding of the invention. These compounds are of a type described in

WO 2014/063054.

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). Table 1 - Compounds

Synthetic Procedure

The synthesis of the compounds of the invention was devised using the synthetic pathway developed for the known inhibitor BMX-IN-1 (see Liu et al. ACS Chem. Biol. 2013).

However, some modifications were introduced, either to improve the yield of intermediate steps or because the reported procedure failed in our hands (see Scheme 1).

Scheme 1 - Synthetic route for the preparation of compounds 9a-e, 10-13 and 14-23.

a) Reflux, 24 h; Ph₂0, 240°C, 5h, 40%; b) SOCI₂, reflux, 5h, 100%; c) aniline, dioxane, 90°C, 6 h, 90%; d) NaBH4, EtOH, r.t., overnight, 100%; e) Dess-Martin Periodinane, DCM, r.t., 3 h, 75%; f) Triethylphosphonoacetate, K2CO3, EtOH, 100°C, overnight, 60%; g) boronic acid/ester, PdCl₂(PPti3)₂, Na2C03, dioxane, 90°C, overnight, 50% or amine, Pd(OAc)2, R-BINAP, CS2CO3, dioxane, 85°C, overnight, 80%; h) Fe, NH₄CI, EtOH/H₂0, 80°C, 2 h, 100%; i) acyl chloride, DIPEA, THF, -10°C to r.t. 5 h, 25% or alkyl halide, K₂CO3, DMF, r.t., overnight, 35%.

Briefly, the formation of the bromohydroxyl quinoline 1 was achieved through a two-step reaction. First, the reaction between 4-bromoaniline and the malonate is carried out at ca. 140-150°C overnight and the cyclization is carried out in diphenyl ether at 225°C overnight. Despite several reports in the literature using a wide range of temperature values

(Price et al.] Ramsey et a /.; Lin et al.] Reis et al.), it was found that when employing gram amounts of starting materials, a strict control of the temperature between 230°C and 240°C was crucial to obtain the product in high yields (Rivilli et ai). From 1 , intermediate 2 was achieved with 100% yield by refluxing in SOC .

Preparation of anilines 3 was accomplished via nucleophilic aromatic substitution with the required aniline. While the reduction of the ester 3 to alcohol 4 was achieved with NaBH₄, the oxidation to aldehyde 5 could not be performed with MnC>2 as originally described (Liu et at. ACS Chem. Biol. 2013). The oxidation could be achieved with Pyridinium chlorochromate (PCC) with NaOAc, however, using Dess-Martin Periodinane (DMP) the oxidation was smoother with less side products. Therefore, the alcohols 4 were oxidized with DMP originating the aldehydes 5. The Horner-Wadsworth-Emmons (HWE) cyclization with triethylphosphonoacetate afforded intermediates 6.

In the next step, 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. Finally, 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). Instead, a more environmentally-friendly reagent was employed (Fe/NFUCI in boiling EtOH) affording intermediates 8 in nearly quantitative yields. Final compounds 9-23 were obtained by acylation of intermediate 8 with the corresponding acyl chloride or by nucleophilic substitution with 4-bromobut-2-enetrile and methyl 4-bromocrotonate to afford 12 and 13, respectively. Analogues 24-27 were prepared in the same way, using

3-bromoaniline as starting material (Scheme 2).

Scheme 2 - Synthetic route for the preparation of compounds 24-27

a) Reflux 24h; RIΊSO, 240°C, 5h, 40%; b) SOCI2, reflux, 5h, 100%; c) aniline, dioxane, 90°C, overnight, 80%; d) NaBFU, EtOH, r.t., ovn, 90%; e) Dess-Martin Periodinane, DCM, r.t., 3h, 80%; f)

Triethylphosphonoacetate, K2CO3, EtOH, 100°C, overnight, 70%; g) boronic acid/ester, PdCl₂(PPh3)₂, Na2C03, dioxane, 90°C, ovn, 20% or amine, Pd(OAc)2, R-BINAP, CS2CO3, dioxane, 85°C, overnight, 90%; h) Fe, NH₄CI, Et0H/H₂0, 80°C, 2h, 70%; i) acryloyl chloride, DIPEA, THF, -10°C to r.t. 5h, 15%. In order to prepare compound 28, first a Suzuki coupling between intermediate 3a and the boronic ester was performed under the described conditions (see conditions g) in

Scheme 1), followed by reduction of the nitro group with Fe/NH4CI and acylation with acryloyl chloride (Scheme 3). Finally, compound 29 was prepared from intermediate 6a which was reduced with SnCh, followed by acylation with acryloyl chloride (Scheme 4).

Scheme 3 - Synthetic route for the preparation of compound 28

a) 4-(methanesulfonylamino) phenyl boronic acid pinacol ester, PdCl₂(PPh3)₂, Na₂CC>3, dioxane, 90°C, overnight, 73%; b) Fe, NhUCI, EtOH/hhO, 80°C, 2h, 88%; c) acryloyl chloride, DIPEA, THF, -10°C to RT, 5h, 10%. Scheme 4 - Synthetic route for the preparation of compound 29

a) SnCI₂, EtOAc, 85°C, 2 h, 68%; b) acyl chloride, DIPEA, THF, -10°C to RT, 5h, 95%.

6-Bromo-4-hydroxyquinoline-3-carboxylate (1)

Diethyl 2-(ethoxymethylene) malonate (11.7 ml_; 58.13 mmol) and 4-bromoaniline (10 g; 58.13 mmol) were heated to 145°C. After 23 h, the solvent was evaporated affording an off- white solid. Ph₂0 (25 ml_) was added and the reaction heated to 245°C. After 6h, no more intermediate was detected by TLC (EtOAc:Hexane 20:80). Upon cooling to r.t., a precipitate was formed, and hexane was added to induce more precipitation. The precipitate was filtered, washed with EtOAc and dried in vacuum to afford the title compound as an off-white solid (6.9 g; 40% yield).

¹H NMR (300 MHz, D₆-DMSO): d 8.60 (s, 1 H), 8.22 (d, J = 2.4 Hz, 1 H), 7.82 (dd, J = 8.7,

2.4 Hz, 1 H), 7.58 (d, J = 8.7 Hz, 1 H), 4.20 (q, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H).

Ethyl 6-bromo-4-chloro-3-quinolinecarboxylate (2)

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).

¹H NMR (300 MHz, CDCI3): d 9.37 (s, 1 H), 8.71 (d, J = 2.0 Hz, 1 H), 8.56 (d, J = 9.0 Hz, 1 H), 8.12 (dd, J = 9.0, 2.0 Hz, 1 H), 4.53 (q, J = 7.1 Hz, 2H), 1.47 (t, J = 7.1 Hz, 3H). General Procedure A: Nucleophilic Aromatic Substitution

Ethyl 6-bromo-4-((4-methyl-3-nitrophenyl)amino)quinoline-3-carboxylate (3a)

Ethyl 6-bromo-4-chloro-3-quinolinecarboxylate 2 (800 mg; 2.543 mmol) and 4-methyl-5- nitroaniline (387 mg; 2.543 mmol) were mixed in dioxane (15 ml_) and heated to 90°C. After 7 h, TLC analysis (50% EtOAc/Hexane) no longer detected starting materials. The yellow suspension was cooled to r.t., diluted with H₂0 and NaOH (1 M) added until pH = 8 was reached. EtOAc was added and the phases were separated. The aqueous phase was further extracted with EtOAc (2 x) and the combined organics were washed with brine and dried over MgS04. After filtration, the solvent was evaporated to afford the title compound as a bright-yellow solid (980 mg; 90% yield).

¹H NMR (300 MHz, CDCIs): d 10.38 (s, 1 H), 9.29 (s, 1 H), 7.90 (d, J = 9.4 Hz, 1 H), 7.74 (dq, J = 4.3, 2.2 Hz, 2H), 7.65 (d, J = 2.5 Hz, 1 H), 7.25 (d, J = 8.3 Hz, 1 H), 7.08 (dd, J = 8.3,

2.5 Hz, 1 H), 4.45 (q, J = 7.1 Hz, 2H), 2.58 (s, 3H), 1.46 (t, J = 7.1 Hz, 3H).

Ethyl 6-bromo-4-((3-methyl-5-nitrophenyl)amino)quinoline-3-carboxylate (3b)

Prepared using General Procedure A, reacting intermediate 2 with 3-methyl-5-nitroaniline. Compound 3b was isolated as a yellow solid (730 mg; 89% yield).

¹H NMR (300 MHz, CDCIs): d 10.35 (s, 1 H), 9.30 (s, 1 H), 7.92 (d, J = 8.8 Hz, 1 H), 7.79-7.73 (m, 3H), 7.61 (s, 1 H), 7.11 (s, 1 H), 4.45 (q, J = 7.1 Hz, 2H), 2.39 (s, 3H), 1.45 (t, J = 7.1 Hz, 3H). ¹³C NMR (75.5 MHz, CDCIs): d 167.9, 151.5, 150.2, 149.6, 149.0, 143.5, 141.2, 135.1 , 132.1 , 128.4, 127.5, 120.8, 1 19.7, 1 19.2, 1 13.2, 109.1 , 61.9, 14.4. HRMS (ESI): m/z [M + H]+ calc for Ci₉Hi₇BrN₃0₄: 430.0397; found: 430.0401.

Ethyl 6-bromo-4-((2-methyl-5-nitrophenyl)amino)quinoline-3-carboxylate (3c)

Prepared using General Procedure A, reacting intermediate 2 with 2-methyl-5-nitroaniline. The reaction required heating at 90°C during 23 h followed by 4 h at 110°C. Compound 3c was isolated as a yellow solid (730 mg, 89% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.29 (s, 1 H), 8.87 (s, 1 H), 8.52 - 8.47 (m, 1 H), 7.98 - 7.90 (m, 3H), 7.66 (d, J = 2.4 Hz, 1 H), 7.60 (d, J = 8.5 Hz, 1 H), 3.85 (q, J = 7.1 Hz, 2H), 2.44 (s, 3H), 1.04 (t, J = 7.1 Hz, 3H). HRMS (ESI): m/z [M + H]+ calc for Ci9Hi₇BrN₃04: 430.0397; found: 430.0400. ¹³C NMR could not be acquired because the compound is not sufficiently soluble in D₆-DMSO, D₆-acetone, D₃-acetonitrile or D₄-methanol. Ethyl 6-bromo-4-((3-nitrophenyl)amino)quinoline-3-carboxylate (3d)

Prepared using General Procedure A, reacting intermediate 2 with 3-nitroaniline. The reaction time was 8 h. Compound 3d was isolated as an orange solid (640 mg: 81 % yield).

¹H NMR (300 MHz, CDCI₃): d 10.41 (s, 1 H), 9.32 (s, 1 H), 8.01 - 7.91 (m, 2H), 7.86 (t, J = 2.2 Hz, 1 H), 7.79 - 7.70 (m, 2H), 7.51 - 7.42 (m, 1 H), 7.28 - 7.23 (m, 1 H), 4.47 (q, J = 7.1 Hz, 2H), 1.47 (t, J = 7.2 Hz, 3H). 13C NMR (75.5 MHz, CDCI3): d 167.9, 151.5, 150.1 , 149.5, 149.0, 143.7, 135.1 , 132.1 , 130.0, 128.3, 126.6, 120.8, 1 19.4, 119.0, 115.9, 109.4, 62.0,

14.3. HRMS (ESI): m/z [M + H]+ calc for Ci₈Hi₅BrN₃0₄: 416.0240; found: 416.0245.

Ethyl 6-bromo-4-((4-methoxy-3-nitrophenyl)amino)quinoline-3-carboxylate (3e)

Prepared using the General Procedure A, reacting intermediate 2 with 4-methoxy-3- nitroaniline. Compound 3e was isolated as an orange solid (780 mg; 92% yield).

¹H NMR (300 MHz, CDCIs): d 10.43 (s, 1 H), 9.26 (s, 1 H), 7.88 (d, J = 9.3 Hz, 1 H), 7.73 - 7.70 (m, 2H), 7.59 (d, J = 2.7 Hz, 1 H), 7.22 - 7.18 (m, 1 H), 7.04 (d, J = 9.0 Hz, 1 H), 4.45 (q,

J = 7.1 Hz, 2H), 3.98 (s, 3H), 1.46 (t, J = 7.1 Hz, 3H). 13C NMR (75.5 MHz, CDCI3): d 168.2, 151.7, 151.1 , 150.4, 149.7, 139.7, 135.2, 134.9, 132.1 , 128.4, 128.1 , 120.4, 119.7, 118.9, 114.6, 107.8, 61.8, 57.1 , 14.4. HRMS (ESI): m/z [M + H]+ calc for Ci₉Hi₇BrN₃0₅: 446.0346; found: 446.0347.

Ethyl 7-bromo-4-(4-methyl-3-nitrophenylamino)quinoline-3-carboxylate (3a’)

Prepared using General Procedure A, reacting intermediate 2’ with 4-methyl-5-nitroaniline. The reaction was completed after 3 h. Compound 3f was isolated as a yellow solid (1.85 g; 85% yield).

¹H NMR (300 MHz, CDCIs): d 10.54 (s, 1 H), 9.31 (s, 1 H), 8.26 (d, J = 2.0 Hz, 1 H), 7.69 (d, J = 2.4 Hz, 1 H), 7.48 (d, J = 9.1 Hz, 1 H), 7.34 (dd, J = 9.1 , 2.0 Hz, 1 H), 7.28 (m, 1 H), 7.13 (dd, J = 8.3, 2.4 Hz, 1 H), 4.48 (q, J = 7.1 Hz, 2H), 2.60 (s, 3H), 1.49 (t, J = 7.1 Hz, 3H). ¹³C NMR (75.5 MHz, CDCI₃): d 168.0, 152.1 , 151.8, 151.3, 149.6, 141.5, 133.7, 132.4, 129.6, 128.8, 127.3, 126.4, 126.2, 1 17.9, 1 17.7, 108.0, 61.9, 20.1 , 14.4. HRMS (ESI): m/z [M + H]+ calc for Ci₉Hi₇BrN₃0₄: 430.0397; found: 430.0393. Ethyl 7-bromo-4-(3-methyl-5-nitrophenylamino)quinoline-3-carboxylate (3b’)

Prepared using General Procedure A, reacting intermediate 2’ with 3-methyl-5-nitroaniline. Reaction time was 17 h. The crude was washed with cold EtOAc to remove unreacted aniline. Compound 3b’ was isolated as an orange solid (740 mg: 58% yield).

¹H NMR (300 MHz, CDCI₃) d 10.41 (s, 1 H), 9.31 (s, 1 H), 8.35 - 8.08 (m, 1 H), 7.79 (s, 1 H), 7.63 (s, 1 H), 7.46 (d, J = 9.1 Hz, 1 H), 7.37 - 7.28 (m, 1 H), 7.10 (s, 1 H), 4.46 (q, J = 7.0 Hz, 2H), 2.39 (s, 3H), 1.46 (t, J = 7.1 Hz, 3H). HRMS (ESI): m/z [M + H]+ calc for Ci₉Hi₇BrN₃0₄: 430.0397; found: 430.0390.

Ethyl 7-bromo-4-(3-nitrophenylamino)quinoline-3-carboxylate (3d’)

Prepared using General Procedure A, reacting intermediate 2’ with 3-nitroaniline.

Reaction time was 17 h. Compound 3d’ was isolated as an orange solid (2.52 g: 95% yield).

¹H NMR (300 MHz, CDCIs): d 10.41 (s, 1 H), 9.31 (s, 1 H), 8.24 (brs, 1 H), 7.79 (s, 1 H), 7.63 (s, 1 H), 7.46 (d, J = 9.1 Hz, 1 H), 7.34 - 7.28 (m, 1 H), 7.10 (s, 1 H), 4.46 (q, J = 7.1 Hz, 2H), 2.39 (s, 3H), 1.46 (t, J = 7.1 Hz, 3H). ¹³C NMR (75.5 MHz, CDCh): d 168.0, 152.3, 151.7, 151.2, 149.1 , 144.0, 132.9, 130.3, 129.0, 127.2, 126.9, 126.3, 119.0, 118.2, 116.0, 108.8, 62.0, 14.4. HRMS (ESI): m/z [M + H]+ calc for Ci₈Hi₅BrN₃0₄: 416.0240; found: 416.0244.

General Procedure B: Ester to Alcohol Reduction

(6-Bromo-4-((4-methyl-3-nitrophenyl)amino)quinolin-3-yl)methanol (4a)

Sodium borohydride (5.44 g; 143.99 mmol; 15 equiv.) was added portionwise to a stirred solution of 3a (4.13 g; 9.599 mmol) in EtOH (35 ml_) at 0°C. After 15 h, TLC analysis (50% EtOAc/Hexane) showed disappearance of starting material. The orange solution was cooled in an ice-bath and quenched with NH₄CI aq. The mixture was partitioned between H₂0 and EtOAc. The phases were separated and the aqueous phase further extracted with EtOAc (2 x). The combined organics were washed with brine, dried over MgS0₄ and evaporated to dryness to afford the title compound as an orange solid (3.73 g; 100% yield).

(6-Bromo-4-((3-methyl-5-nitrophenyl)amino)quinolin-3-yl)methanol (4b)

Prepared using the procedure described for 4a. Compound 4b was obtained as an orange solid (630 mg; 100%). ¹H NMR (300 MHz, D₆-DMSO): d 9.07 (s, 1 H), 9.00 (s, 1 H), 8.24 (s, 1 H), 8.00 (d, J = 8.9 Hz, 1 H), 7.87 (d, J = 8.9 Hz, 1 H), 7.48 (s, 1 H), 7.28 (s, 1 H), 6.85 (s, 1 H), 5.47 (t, J = 5.4 Hz, 1 H), 4.48, (d, J = 5.4 Hz, 2H), 2.31 (s, 3H). 13C NMR (75.5 MHz, d6-DMS0): d 152.2, 148.6, 146.9, 146.1 , 140.6, 139.9, 132.3, 131.7, 128.7, 125.5, 125.3, 121.3, 119.6, 114.3, 106.5, 58.2, 20.9. HRMS (ESI): m/z [M + H]+ calc for Ci₇Hi₅BrN₃0₃: 388.0291 ; found: 388.0293.

(6-Bromo-4-((2-methyl-5-nitrophenyl)amino)quinolin-3-yl)methanol (4c)

Prepared using the procedure described for 4a. Compound 4c was obtained as an orange solid (250 mg; 68% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.03 (s, 1 H), 8.20 (d, J = 2.1 Hz, 1 H), 8.09 (s, 1 H), 8.00 (d, J = 8.9 Hz, 1 H), 7.87 (dd, J = 8.9, 2.2 Hz, 1 H), 7.68 (dd, J = 8.3, 2.3 Hz, 1 H), 7.50 (dd, J = 8.3, 0.9 Hz, 1 H), 6.88 (d, J = 2.4 Hz, 1 H), 5.47 (t, J = 5.5 Hz, 1 H), 4.39, (d, J = 5.5 Hz, 2H), 2.50 (s, 3H). 13C NMR (75.5 MHz, d6-DMSO): d 152.0, 147.0, 146.5, 144.4, 141.5, 134.5, 132.4, 131.7, 131.5, 128.2, 125.7, 125.2, 1 19.5, 115.0, 109.2, 58.3, 18.4. HRMS (ESI): m/z [M + H]+ calc for CiyHisBrNsOs: 388.0291 ; found: 388.0293.

(6-Bromo-4-((3-nitrophenyl)amino)quinolin-3-yl)methanol (4d)

Prepared using the procedure described for 4a. Purification was carried out by column chromatography over silica-gel (eluent: MeOH:DCM 0:100 to 5:95) to give compound 4d as a yellow solid (250 mg; 42% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.06 (d, J = 5.4 Hz, 2H), 8.21 (d, J = 2.0 Hz, 1 H), 7.99 (d, J = 8.9 Hz, 1 H), 7.87 (d, J = 8.9 Hz, 1 H), 7.64 (d, J = 8.0 Hz, 1 H), 7.49 - 7.42 (m, 2H), 7.00 (d, J = 8.0 Hz, 1 H), 5.47 (t, J = 5.1 Hz, 1 H), 4.48 (d, J = 5.1 Hz, 2H). ¹³C NMR (75.5 MHz, D₆- DMSO): d 152.3, 148.6, 147.0, 146.3, 139.9, 132.4, 131.7, 130.5, 128.8, 125.5, 125.3,

120.9, 1 19.7, 1 13.7, 109.1 , 58.2. HRMS (ESI): m/z [M + H]+ calc for Ci₆Hi₃BrN₃0₃:

374.0135; found: 374.0134.

(6-Bromo-4-((4-methoxy-3-nitrophenyl)amino)quinolin-3-yl)methanol (4e)

Prepared using the procedure described for 4a. Compound 4e was obtained as an orange solid (600 mg; 93% yield)

¹H NMR (300 MHz, D₆-DMSO): d 8.97 (s, 1 H), 8.67 (s, 1 H), 8.26 (d, J = 2.1 Hz, 1 H), 7.94 (d, J = 8.9 Hz, 1 H), 7.83 (dd, J = 8.9, 2.1 Hz, 1 H), 7.32 - 7.18 (m, 2H), 7.00 (dd, J = 9.0, 2.8 Hz, 1 H), 5.40 (t, J = 5.4 Hz, 1 H), 4.42 (d, J = 5.4 Hz, 2H), 3.85 (s, 3H). ¹³C NMR (75.5 MHz, d6- DMSO): d 152.2, 147.0, 146.0, 141.1, 139.3, 138.2, 132.1, 131.7, 131.7, 126.5, 125.5, 124.6, 122.3, 119.2, 115.5, 112.3, 58.5, 56.9. HRMS (ESI): m/z [M + H]+ calc for

CiyHisBrNsCU: 404.0240; found: 404.0244.

(7-Bromo-4-(4-methyl-3-nitrophenylamino)quinolin-3-yl)methanol (4a’)

Prepared using the procedure described for 4a. Compound 4a’ was isolated as an orange solid (770 mg; 100% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.00 (s, 1H), 8.83 (s, 1H), 8.23 (d, J= 2.0 Hz, 1H), 7.88 (d, J= 9.0 Hz, 1H), 7.68 (dd, J= 9.0, 2.1 Hz, 1H), 7.32-7.21 (m, 2H), 6.86 (dd, J= 8.4, 2.5 Hz, 1H), 5.43 (t, J= 5.4 Hz, 1H), 4.51 (d, J= 5.4 Hz, 2H), 2.39 (s, 3H). ¹³C NMR (75.5 MHz, De-DMSO): d 152.9, 149.2, 149.1, 144.2, 141.7, 133.4, 131.3, 129.2, 127.8, 125.8, 123.0, 122.5, 122.4, 120.2, 110.6, 58.3, 18.9. HRMS (ESI): m/z [M + H]+ calc for Ci₇Hi₅BrN₃0₃: 388.0291; found: 388.0293.

(7-Bromo-4-(3-methyl-5-nitrophenylamino)quinolin-3-yl)methanol (4b’)

Prepared using the procedure described for 4a. Compound 4b’ was isolated as a yellow solid (620 g; 96% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.03 (s, 1H), 8.94 (s, 1H), 8.24 (d, J= 2.0 Hz, 1H), 7.88 (d, J = 9.0 Hz, 1 H), 7.68 (dd, J = 9.0, 2.0 Hz, 1 H), 7.48 - 7.45 (m, 1 H), 7.25 (m, 1 H), 6.84 (brs, 1H), 5.47 (t, J= 5.4 Hz, 1H), 4.52 (d, J= 5.4 Hz, 2H), 2.29 (s, 3H). HRMS (ESI): m/z

[M + H]+ calc for CiyHisBrNsOs: 388.0291; found: 388.0294.

(7-Bromo-4-(3-nitrophenylamino)quinolin-3-yl)methanol (4d’)

Prepared using the procedure described for 4a. Compound 4d’ was isolated as an orange solid (810 mg; 91% yield).

¹H NMR (300 MHz, De-DMSO): d 9.03 (s, 2H), 8.25 (d, J = 2.0 Hz, 1 H), 7.87 (d, J = 9.0 Hz, 1H), 7.69 (dd, J= 9.0, 2.1 Hz, 1H), 7.62 (ddd, = 8.1, 2.2, 0.9 Hz, 1H), 7.48-7.44 (m, 1H), 7.41 (d, J= 8.1 Hz, 1 H), 5.82 (s, 1H), 5.51 (t, J= 5.5 Hz, 1H), 4.52 (d, J= 5.5 Hz, 2H).

HRMS (ESI): m/z [M + H]+ calc for Ci₆Hi₃BrN₃0₃: 374.0135; found: 374.0134. General Procedure C: Alcohol Oxidation to Aldehyde

6-Bromo-4-((4-methyl-3-nitrophenyl)amino)quinoline-3-carbaldehyde (5a)

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.. H₂0 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).

6-Bromo-4-((3-methyl-5-nitrophenyl)amino)quinoline-3-carbaldehyde (5b)

Prepared using the procedure described for 5a. Compound 5b was obtained as an orange solid (420 g; 72% yield).

¹H NMR (300 MHz, CDCIs): d 1 1.26 (s, 1 H), 10.10 (s, 1 H), 8.90 (s, 1 H), 7.97-7.88 (m, 2H), 7.83-7.74 (m, 2H), 7.66 (d, J = 2.1 Hz, 1 H), 7.34-7.27 (m, 1 H), 2.46 (s, 3H). ¹³C NMR (75.5 MHz, CDCI3): d 193.4, 155.0, 150.2, 149.7, 149.0, 141.6 (2), 136.0, 132.2, 129.5, 128.7, 121.4, 1 19.2, 1 18.9, 1 15.3, 1 13.7, 21.5. HRMS (ESI): m/z [M + H]+ calc for

CiyHisBrNsOs: 386.0135; found: 386.0142.

6-Bromo-4-((2-methyl-5-nitrophenyl)amino)quinoline-3-carbaldehyde (5c)

Prepared using the procedure described for 5a. Compound 5c was obtained as a yellow solid (165 mg; 72% yield).

¹H NMR (300 MHz, D₆-DMSO): d 10.18 (s, 1 H), 9.94 (s, 1 H), 8.97 (s, 1 H), 8.07-8.02 (m, 2H), 7.93-7.88 (m, 3H), 7.67 (m, 1 H), 2.42 (s, 3H). ¹H NMR (300 MHz, CDCI₃): d 11.28 (s, 1 H), 10.11 (s, 1 H), 8.90 (s, 1 H), 8.1 1 (dd, J = 8.4, 2.1 Hz, 1 H), 7.91 (d, J = 8.9 Hz, 2H), 7.75 (dd,

J = 9.0, 2.1 Hz, 1 H), 7.55 (d, J = 8.4 Hz, 1 H), 7.45 (d, J = 1.9 Hz, 1 H), 2.48 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for CiyHnBrNsOs: 386.0135; found: 386.0136.

6-Bromo-4-((3-nitrophenyl)amino)quinoline-3-carbaldehyde (5d)

Prepared using the procedure described for 5a. Compound 5d was obtained as an orange solid (200 mg; 65% yield). ¹H NMR (300 MHz, CDCIs): d 11.27 (s, 1H), 10.12 (s, 1H), 8.93 (s, 1H), 8.14 (dd, J = 8.1, 2.2 Hz, 1 H), 8.03 (t, J =2.2 Hz, 1H), 7.93 (d, J = 9.0 Hz, 1H), 7.78 (dd, J = 9.0, 2.1 Hz, 1H), 7.64 (d, J = 2.1 Hz, 1 H), 7.60-7.53 (m, 1H), 7.45 (d, J = 8.0 Hz, 1H). ¹³C NMR (75.5 MHz,

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. HRMS (ESI): m/z [M + H]+ calc for CieHuBrNsCh: 371.9978; found: 371.9986.

6-Bromo-4-((4-methoxy-3-nitrophenyl)amino)quinoline-3-carbaldehyde (5e)

Prepared using the procedure described for 5a. Compound 5e was obtained as an orange solid (370 g; 70% yield). ¹H NMR (300 MHz, CDCIs): d 11.33 (s, 1H), 10.07 (s, 1H), 8.84 (s, 1H), 7.88 (dd, J = 8.9, 2.1 Hz, 1H), 7.77-7.70 (m, 2H), 7.63 (d, J = 2.1 Hz, 1H), 7.38 (dd, J = 8.8, 2.7 Hz, 1H), 7.14 (d, J = 8.9 Hz, 1H), 4.02 (s, 3H).13C NMR (75.5 MHz, CDCI3): d 193.3, 155.2, 151.6, 151.0, 149.7, 139.7, 135.8, 133.0, 132.1, 129.9, 128.7, 121.5, 119.0, 118.8, 114.8, 112.9, 57.1. HRMS (ESI): m/z [M + H]+ calc for CiyH BrNsCU: 402.0084; found: 402.0088.

7-Bromo-4-(4-methyl-3-nitrophenylamino)quinoline-3-carbaldehyde (5a’)

Prepared using the procedure described for 5a. Compound 5a’ was obtained as a brown solid (220 mg; 85% yield).

¹H NMR (300 MHz, CDCIs): d 11.29 (s, 1H), 10.06 (s, 1H), 8.85 (s, 1H), 8.18 (d, J = 2.0 Hz, 1 H), 7.80 (d, J = 2.4 Hz, 1 H), 7.40 - 7.32 (m, 2H), 7.30 - 7.24 (m, 2H), 2.62 (s, 3H).

¹³C NMR (75.5 MHz, CDCIs): d 193.3, 155.9, 151.8, 151.6, 149.7, 139.8, 134.1, 132.9, 131.4, 128.8, 127.9, 127.5, 127.4, 119.5, 116.6, 113.3, 20.2. HRMS (ESI): m/z [M + H]+ calc. for Ci₇Hi₃BrN₃0s: 386.0135; found: 386.0135.

7-Bromo-4-(3-methyl-5-nitrophenylamino)quinoline-3-carbaldehyde (5b’)

Prepared using the procedure described for 5a. Compound 5b’ was obtained as an orange solid (480 mg; 82% yield).

¹H NMR (300 MHz, D₆-DMSO): d 10.37 (s, 1H), 10.11 (s, 1H), 9.03 (s, 1H), 8.22 (d, J= 2.1 Hz, 1 H), 7.92 (d, J= 9.1 Hz, 1H), 7.78-7.75 (m, 2H), 7.67 (dd, J= 9.0, 2.1 Hz, 1H), 7.38 (s, 1H), 2.34 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for CiyHisBrNsCh: 386.0135; found: 386.0127. 7-Bromo-4-(3-nitrophenylamino)quinoline-3-carbaldehyde (5d’)

Prepared using the procedure described for 5a. Compound 5d’ was obtained as a brown solid (790 mg; 99% yield).

¹H NMR (300 MHz, CDCI₃): d 1 1.32 (s, 1 H), 10.10 (s, 1 H), 8.92 (s, 1 H), 8.23 (d, J = 2.0 Hz, 1 H), 8.17 - 8.05 (m, 1 H), 8.03 - 8.01 (m, 1 H), 7.55 (t, J = 8.1 Hz, 1 H), 7.50 - 7.41 (m, 1 H), 7.38 (d, J = 9.1 Hz, 1 H), 7.29 (dd, J = 9.1 , 2.0 Hz, 1 H). HRMS (ESI): m/z [M + H]+ calc for CieHuBrNsOs: 371.9978; found: 371.9975.

General Procedure D: Horner-Wadsworth-Emmons (HWE) cyclization

9-Bromo-1-(4-methyl-3-nitrophenyl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (6a)

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).

9-Bromo-1-(3-methyl-5-nitrophenyl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (6b)

Prepared using the procedure described for 6a. Compound 6b was obtained as a brown solid (320 mg; 74% yield).

¹H NMR (300 MHz, CDCI₃): d 8.97 (s, 1 H), 8.33 (s, 1 H), 8.09 - 7.88 (m, 3H), 7.67 (dd, J = 8.9, 2.1 Hz, 1 H), 7.53 (s, 1 H), 6.95 (d, J = 9.5 Hz, 1 H), 6.80 (d, J = 2.1 Hz, 1 H), 2.58 (s, 3H). ¹³C NMR (75.5 MHz, CDCI3): d 162.9, 150.8, 149.4, 148.2, 142.7, 141.2, 140.6, 139.7, 135.6, 133.5, 132.6, 127.5, 125.1 , 122.9, 121.4, 120.3, 118.44, 1 13.8, 21.5. HRMS (ESI): m/z [M + H]+ calc for DigHisBrNsOs: 410.0135; found: 410.0132.

9-Bromo-1-(2-methyl-5-nitrophenyl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (6c)

Prepared using the procedure described for 6a. Compound 6c was obtained as a dark brown solid (165 mg; 68% yield). ¹H NMR (300 MHz, CDCI₃): d 9.02 (s, 1H), 8.45 (dd, J= 8.5, 2.4 Hz, 1H), 8.11 -7.97 (m, 3H), 7.74-7.68 (m, 2H), 7.00 (d, J= 9.5 Hz, 1H), 6.80 (d, J= 2.0 Hz, 1H), 2.21 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for Ci₉Hi₃BrN₃0₃: 410.0135; found: 410.0133.

9-Bromo-1-(3-nitrophenyl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (6d)

Prepared using the procedure described for 6a. Compound 6d was obtained as a dark brown solid (150 mg; 71% yield).

¹H NMR (300 MHz, CDCIs): d 9.01 (s, 1H), 8.53 (dd, J= 8.3, 2.1 Hz, 1H), 8.21 (t, J= 2.1 Hz, 1H), 8.00 (dd, J= 10.4, 9.2 Hz, 2H), 7.87 (t, J= 8.1 Hz, 1H), 7.80- 7.64 (m, 2H), 6.97 (d, J = 9.5 Hz, 1H), 6.80 (d, J=2.1 Hz, 1H). HRMS (ESI): m/z [M + H]+ calc for Ci₈HiiBrN₃0₃: 395.9978; found: 395.9976.

9-Bromo-1-(4-methoxy-3-nitrophenyl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (6e)

Prepared using the procedure described for 6a. Compound 6e was obtained as a dark brown solid (255 g; 65% yield)

¹H NMR (300 MHz, D₆-DMSO): 69.17 (s, 1H), 8.32 (d, J= 9.5 Hz, 1H), 8.17 (d, J = 2.5 Hz, 1H), 7.98 (d, J= 8.9 Hz, 1H), 7.82 (m, 2H), 7.69 (d, J= 9.0 Hz, 1H), 6.96 (d, J= 9.4 Hz, 1H), 6.83 (d, J = 2.0 Hz, 1 H), 4.07 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for Ci₉Hi₃BrN₃0₄: 426.0084; found: 426.0090.

8-Bromo-1-(4-methyl-3-nitrophenyl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (6a’)

Prepared using the procedure described for 6a. Compound 6a’ was obtained as a brown solid (220 mg; 96% yield).

¹H NMR (300 MHz, D₆-DMSO): 69.17 (s, 1H), 8.31 (d, J= 9.5 Hz, 1H), 8.23 (m, 2H), 7.81 - 7.68 (m, 2H), 7.39 (dd, J= 9.4, 2.3 Hz, 1H), 6.94 (d, J= 9.5 Hz, 1H), 6.73 (d, J= 9.4 Hz,

1H), 2.68 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for Ci₉Hi₃BrN₃0₃: 410.0135; found:

410.0134. 8-Bromo-1-(3-methyl-5-nitrophenyl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (6b’)

Prepared using the procedure described for 6a. Compound 6b’ was obtained as a brown solid (345 mg; 81 % yield).

¹H NMR (300 MHz, D₆-DMSO): 6 9.18 (s, 1 H), 8.36 - 8.27 (m, 3H), 8.24 (d, J = 2.3 Hz, 1 H), 7.76 (brs, 1 H), 7.36 (dd, J = 9.5, 2.3 Hz, 1 H), 6.95 (d, J = 9.4 Hz, 1 H), 6.69 (d, J = 9.4 Hz, 1 H), 2.48 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for CigHisBrNsOs: 410.0135; found: 410.0132.

8-Bromo-1-(3-nitrophenyl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (6d’)

Prepared using the procedure described for 6a. Compound 6d’ was obtained as a brown solid (500 g; 60% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.18 (s, 1 H), 8.49 (m, 2H), 8.32 (d, J = 9.5 Hz, 1 H), 8.24 (d, J = 2.3 Hz, 1 H), 7.91 - 7.89 (m, 2H), 7.34 (dd, J = 9.4, 2.3 Hz, 1 H), 6.95 (d, J = 9.5 Hz, 1 H), 6.65 (d, J = 9.4 Hz, 1 H). HRMS (ESI): m/z [M + H]+ calc for CisHu BrNsOs: 395.9978; found: 395.9975.

General Procedure E: Suzuki Coupling

N-(4-(1-(4-Methyl-3-nitrophenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin-9- yl)phenyl)methanesulfonamide (7a)

The bromo-quinoline 6a (280 mg; 0.683 mmol), 4-(methanesulfonylamino) phenyl boronic acid pinacol ester (243 mg; 0.819 mmol; 1.2 equiv.), PdCl2(PPh3)2 (48 mg; 0.068 mmol;

0.1 equiv.) and Na₂CC>3 (1.025 ml; 2 M, 2.049 mmol; 3 equiv.) were mixed in dioxane (3 ml_) under Argon. The mixture was heated to 90°C overnight. After 16 h, TLC analysis

(MeOH:DCM 5:95) showed that the reaction was completed. The mixture was cooled to RT and filtered through a Celite pad. The pad was further washed with EtOH and MeOH/DCM (10%) until no more product was detected by TLC. The solvent was evaporated and the crude applied in a silica column with a gradient up to 2% MeOH in DCM. The desired fractions were collected and evaporated to dryness to afford the title compound as a yellow solid (455 mg; 69%). N-(4-(1-(3-Methyl-5-nitrophenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9- yl)phenyl)methanesulfonamide (7b)

Prepared using the procedure described for 7a. Compound 7b was obtained as a dark yellow solid (160 mg: 64% yield).

¹H NMR (300 MHz, D₆-DMSO): 69.92 (brs, 1H), 9.15 (s, 1H), 8.45-8.28 (m, 3H), 8.10 (d, J = 8.6 Hz, 1 H), 7.98 (dd, J= 8.7, 1.9 Hz, 1H), 7.89 (s, 1H), 7.20 (d, J= 8.6 Hz, 2H), 7.15- 7.02 (m, 3H), 6.95 (d, J= 9.5 Hz, 1H), 3.03 (s, 3H), 2.5 (s, 3H). ¹H NMR (300 MHz, CDCI₃): 69.01 (s, 1H), 8.29 (s, 1H), 8.19 (d, J = 8.7 Hz, 1H), 8.09 (m, 1H), 8.03 (d, J=9.5 Hz, 1H), 7.86 (dd, J= 8.7, 1.9 Hz, 1H), 7.59 (s, 1H), 7.19 (d, J= 8.6 Hz, 2H), 7.12 (d, J= 1.9 Hz, 1H), 7.04 (d, J= 8.6 Hz, 2H), 6.96 (d, J= 9.4 Hz, 1H), 6.57 (s, 1H), 3.07 (s, 4H), 2.56 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₆H₂₁N₄O₅S: 501.1227; found: 501.1224.

N-(4-(1-(2-Methyl-5-nitrophenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9- yl)phenyl)methanesulfonamide (7c)

Prepared using the procedure described for 7a. Compound 7c was obtained as an orange solid (145 g; 77% yield).

¹H NMR (300 MHz, De-acetone): 69.14 (s, 1H), 8.74 (brs, 1H), 8.53-8.44 (m, 2H), 8.34 (d, J= 9.5 Hz, 1H), 8.15 (d, J= 8.6 Hz, 1H), 8.03-7.91 (m, 2H), 7.38-7.31 (m, 2H), 7.23- 7.13 (m, 3H), 6.95 (d, J= 9.5 Hz, 1H), 3.04 (s, 3H), 2.23 (s, 3H). HRMS (ESI): m/z [M + H]+ calc. for C₂₆H₂₁N₄O₅S: 501.1227; found: 501.1223.

N-(4-(1-(3-Nitrophenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9- yl)phenyl)methanesulfonamide (7d)

Prepared using the procedure described for 7a. Compound 7d was obtained as a yellow solid (85 mg; 51% yield).

¹H NMR (300 MHz, D₆-DMSO): 69.93 (s, 1H), 9.15 (s, 1H), 8.62 (s, 1H), 8.58-8.47 (m,

1H), 8.34 (d, J= 9.5 Hz, 1H), 8.10 (d, J= 8.7 Hz, 1H), 7.97 (m, 3H), 7.18 (d, J= 8.2 Hz, 2H), 7.10-6.99 (m, 3H), 6.96 (d, J= 9.4 Hz, 1H), 3.03 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₅H₁₉N₄O₅S: 487.1071; found: 487.1071. N-(4-(1-(4-Methoxy-3-nitrophenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9- yl)phenyl)methanesulfonamide (7e)

Prepared using the procedure described for 7a. Compound 7e was obtained as a dark yellow solid (120 mg; 42% yield).

¹H NMR (300 MHz, D₆-acetone): d 9.06 (s, 1H), 8.83-8.74 (brs, 1H), 8.24 (d, J= 9.5 Hz, 1H), 8.15-8.09 (m, 2H), 7.99 (dd, J= 8.7, 1.9 Hz, 1H), 7.82 (dd, J= 8.9, 2.6 Hz, 1H), 7.64 (d, J= 9.0 Hz, 1 H), 7.40 (d, J= 8.6 Hz, 2H), 7.30 (d, J= 2.2 Hz, 1H), 7.27 (d, J= 2.1 Hz,

1H), 7.21 (dd, J= 1.9, 0.6 Hz, 1H), 6.88 (d, J= 9.5 Hz, 1H), 4.14 (s, 3H), 3.06 (d, J= 0.9 Hz, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₆H₂₁N₄O₆S: 517.1176; found: 517.1176.

N-(4-(1-(4-Methyl-3-nitrophenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-8- yl)phenyl)methanesulfonamide (7a’)

Prepared using the procedure described for 7a. Compound 7a’ was obtained as an orange solid (76 g; 21%).

¹H NMR (300 MHz, D₆-DMSO): d 9.97 (s, 1H), 9.16 (s, 1H), 8.33-8.30 (m, 2H), 8.26 (s,

1 H), 7.84 - 7.77 (m, 4H), 7.57 (dd, J = 9.3, 2.2 Hz, 1 H), 7.30 (d, J = 8.7 Hz, 2H), 6.92 - 6.86 (m, 2H), 3.04 (s, 3H), 2.69 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₆H₂₁N₄O₅S:

501.1227; found: 501.1227.

N-(4-(1-(3-Methyl-5-nitrophenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-8- yl)phenyl)methanesulfonamide (7b’)

Prepared using the procedure described for 7a. Compound 7b’ was obtained as an orange solid (400 mg; 95%).

HRMS (ESI): m/z [M + H]+ calc for C₂₆H₂₁N₄O₅S: 501.1227; found: 501.1232.

N-(4-(1-(3-Nitrophenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-8- yl)phenyl)methanesulfonamide (7d’)

Prepared using the procedure described for 7a. Compound 7d’ was obtained as an orange solid (90 mg; 31%).

¹H NMR (300 MHz, D₆-DMSO): d 9.92 (brs, 1H), 9.16 (s, 1H), 8.52-8.49 (m, 2H), 8.34- 8.29 (m, 2H), 7.95-7.93 (m, 2H), 7.79 (d, J= 8.8 Hz, 2H), 7.52 (dd, J= 9.3, 2.2 Hz, 1H), 7.29 (d, J= 8.8 Hz, 2H), 6.91 (d, J= 9.4 Hz, 1H), 6.76 (d, J= 9.3 Hz, 1H), 3.03 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₅H₁₉N₄O₅S: 487.1071; found: 487.1069.

Ethyl 4-(4-methyl-3-nitrophenylamino)-6-(4-(methylsulfonamido)phenyl)quinoline-3- carboxylate (Intermediate 1 - 1 NT 1)

Prepared using the procedure described for 7a, from intermediate 3a. Compound INT1 was obtained as a yellow solid (265 mg; 73%).

¹H NMR (300 MHz, CDCIs): d 10.52 (s, 1H), 9.29 (s, 1H), 8.10 (d, J= 8.7 Hz, 1H), 7.87 (dd, J = 8.7, 2.0 Hz, 1H), 7.72 (d, J = 2.0Hz, 1H), 7.67 (d, J = 2.5 Hz, 1 H), 7.59 - 7.41 (m, 2H), 7.20-7.16 (m, 5H), 4.46 (q, J= 7.1 Hz, 2H), 3.03 (s, 3H), 2.58 (s, 3H), 1.47 (t, J= 7.1 Hz, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₆H₂₅N₄O₆S: 521.1489; found: 521.1486.

N-(4-(1-(4-Methyl-3-nitrophenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9- yl)phenyl)butane-1 -sulfonamide (7f - precursor of 14)

Prepared using General Procedure E, reacting intermediate 6a with

4-(butylsulfonamido)phenylboronic acid. Compound 7f was isolated as a yellow solid (200 mg; 53% yield)

¹H NMR (300 MHz, D₆-DMSO): d 9.98 (s, 1H), 9.13 (s, 1H), 8.44-8.26 (m, 2H),

8.09 (d, J= 8.7 Hz, 1H), 7.98 (d, J= 8.6 Hz, 1H), 7.82-7.74 (m, 2H), 7.24 (d, J= 8.2 Hz, 2H), 7.09 (d, J= 8.2 Hz, 2H), 6.95 (d, J= 8.2 Hz, 2H), 3.12 (t, J= 7.9 Hz, 3H), 2.65 (s, 3H), 1.65 (m, 2H), 1.36 (m, 2H), 0.84 (t, J= 7.2 Hz, 3H). HRMS (ESI): m/z [M + H]+ calc for

C₂₉H₂₇N₄O₅S: 543.1697; found: 543.1694.

N-(3-(1-(4-Methyl-3-nitrophenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9- yl)phenyl)methanesulfonamide (7g - precursor of 15)

Prepared using General Procedure E, reacting intermediate 6a with

3-(methanesulfonylamino)phenylboronic acid pinacol ester. Compound 7g was isolated as an orange solid (235 mg; 67% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.81 (s, 1H), 9.16 (s, 1H), 8.34 (d, J= 9.5 Hz, 1H), 8.29 (m, 1H), 8.15 (d, J= 8.6 Hz, 1H), 7.91 (dd, J= 8.7, 1.9 Hz, 1H), 7.79 (d, J= 1.3 Hz, 2H),

7.35 (t, J= 7.8 Hz, 1 H), 7.27-7.15 (m, 2H), 7.05 (d, J= 1.8 Hz, 1H), 6.95 (d, J= 9.4 Hz,

1 H), 6.58 (dt, J = 8.0, 1.2 Hz, 1 H), 3.00 (s, 3H), 2.66 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₆H₂₁N₄O₅S: 501.1227; found: 501.1228. Methyl 4-(1-(4-methyl-3-nitrophenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin-9- yl)phenylcarbamate (7h - precursor of 16)

Prepared using General Procedure E, reacting intermediate 6a with

4-(methoxycarbonylamino)benzeneboronic acid. Compound 7h was isolated as an orange solid (166 mg; 50% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.82 (s, 1 H), 9.13 (s, 1 H), 8.33 (dd, J = 9.4, 1.5 Hz, 2H), 8.08 (dd, J = 8.6, 1.4 Hz, 1 H), 8.04 - 7.93 (m, 1 H), 7.86 - 7.72 (m, 2H), 7.56 - 7.46 (m, 2H), 7.06 (dd, J = 8.7, 1.5 Hz, 2H), 7.03 - 6.91 (m, 2H), 3.69 (s, 3H), 2.68 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C27H21N4O5: 481.1506; found: 481.1507.

1-(4-Methyl-3-nitrophenyl)-9-(pyridin-4-yl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (7i -precursor of 17)

Prepared using General Procedure E, reacting intermediate 6a with pyridine-4-boronic acid hydrate. Compound 7i was isolated as an orange solid (90 mg; 45% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.21 (s, 1 H), 8.64 - 8.56 (m, 2H), 8.40 - 8.31 (m, 2H), 8.20 - 8.14 (m, 1 H), 8.1 1 (dd, J = 8.7, 1.9 Hz, 1 H), 7.87 - 7.75 (m, 3H), 7.15 - 7.12 (m, 2H), 6.98 (d, J = 9.5 Hz, 1 H), 2.66 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C24H17N4O3: 409.1295; found: 409.1293.

5-(1-(4-Methyl-3-nitrophenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin-9-yl)picolinonitrile (7j - precursor of 18)

Prepared using General Procedure E, reacting intermediate 6a with 6-(cyanopyridin-3- yl)boronic acid. Compound 7j was isolated as an orange solid (92 mg; 44% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.22 (s, 1 H), 8.50 (m, 1 H), 8.36 (d, J = 9.5 Hz, 1 H), 8.29 (s, 1 H), 8.20 (d, J = 8.6 Hz, 1 H), 8.16 - 8.14 (m, 1 H), 7.80 (m, 2H), 7.65 - 7.52 (m, 3H), 6.98 (d, J = 9.5 Hz, 1 H), 2.66 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C25H16N5O3: 434.1248; found: 434.1245. 1-(4-Methyl-3-nitrophenyl)-9-(6-(trifluoromethyl)pyridin-3-yl)benzo[h][1 ,6]naphthyridin-2(1 H)- one (7k - precursor of 19)

Prepared using General Procedure E, reacting intermediate 6a with 2-trifluoromethyl(pyridin- 5-yl)boronic acid. Compound 7k was isolated as an orange solid (1 10 mg; 47% yield).

¹H NMR (300 MHz, D6-DMSO): d 9.22 (s, 1 H), 8.53 (d, J = 2.1 Hz, 1 H), 8.36 (d, J = 9.5 Hz, 1 H), 8.29 (s, 1 H), 8.20 (d, J = 8.7 Hz, 1 H), 8.13 (dd, J = 8.7, 1.9 Hz, 1 H), 7.95 (d, J = 8.2 Hz, 1 H), 7.83 (m, 1 H), 7.65 - 7.54 (m, 3H), 6.97 (s, 1 H), 2.62 (s, 3H). HRMS (ESI): m/z [M + H]+ calc. for C₂₅H₁₆F₃N₄O₃: 477.1 169; found: 477.1 168.

General Procedure F: Buchwald-Hartwig Coupling

1-(4-Methyl-3-nitrophenyl)-9-(piperidin-1-yl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (7I - precursor of 20)

The bromo-quinoline 6a (250 mg; 0.609 mmol), piperidine (180 pl_; 1.827 mmol; 3 equiv.), Pd(OAc)₂ (8.4 mg; 37.4 pmol; 0.06 equiv.), R-BINAP (46 mg: 73.1 pmol; 0.12 equiv.) and CS₂CO₃ (595 mg, 1.827 mmol; 3 equiv.) were mixed in dioxane (5 ml_) under Argon. The mixture was heated to 90°C overnight. After 24 h, LCMS analysis showed that the reaction was completed. The mixture was cooled to r.t. and the solvent evaporated. The crude was partitioned between EtOAc and sat NaHCOs. The phases were separated and the aqueous phase further extracted with EtOAc (2 x). The combined organics were washed with brine and dried over MgS0₄. After filtration, the solvent was evaporated to afford the title compound as a yellow solid. The compound was used without further purification (180 mg; 71 % yield).

¹H NMR (300 MHz, D₆-DMSO): d 8.87 (s, 1 H), 8.26 - 8.18 (m, 2H), 7.84 (d, J = 9.2 Hz, 1 H), 7.76 (d, J = 8.2 Hz, 1 H), 7.70 (dd, J = 8.2, 2.1 Hz, 1 H), 7.50 - 7.46 (m, 1 H), 6.85 (d, J = 9.4 Hz, 1 H), 6.23 (d, J = 2.6 Hz, 1 H), 2.74 (m, 4H), 2.60 (s, 3H), 1.45 (m, 6H). HRMS (ESI): m/z [M + H]+ calc for C₂₄H₂₃N₄O₃: 415.1765; found: 415.1763.

1-(4-Methyl-3-nitrophenyl)-9-morpholinobenzo[h][1 ,6]naphthyridin-2(1 H)-one (7m -precursor of 21)

Prepared using General Procedure F, using morpholine as amine. Compound 7m was isolated as an orange solid (290 mg; 95% yield).

¹H NMR (300 MHz, D₆-DMSO): d 8.91 (s, 1 H), 8.35 - 8.17 (m, 2H), 7.89 (d, J = 9.2 Hz, 1 H), 7.81 - 7.65 (m, 2H), 7.53 (dd, J = 9.2, 2.6 Hz, 1 H), 6.87 (d, J = 9.4 Hz, 1 H), 6.23 (d, J = 2.6 Hz, 1 H), 3.62 (m, 4H), 2.68 (m, 4H), 2.58 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₃H₂₁N₄O₄: 417.1557; found: 417.1557.

1-(4-Methyl-3-nitrophenyl)-9-(4-(methylsulfonyl)piperazin-1-yl)benzo[h][1 ,6]naphthyridin- 2(1 H)-one (7n - precursor of 22)

Prepared using General Procedure F, using 1-(methylsulfonyl)piperazine as amine.

Compound 7n was isolated as a yellow solid (285 mg; 95% yield).

¹H NMR (300 MHz, CDCIs): d 8.83 (s, 1 H), 8.10 - 7.86 (m, 3H), 7.69 - 7.52 (m, 2H), 7.35 (dd, J = 9.3, 2.6 Hz, 1 H), 6.90 (d, J = 9.3 Hz, 1 H), 6.36 (d, J = 2.6 Hz, 1 H), 3.25 (t, J = 5.0 Hz, 4H), 2.95 - 2.89 (m, 2H), 2.85 - 2.82 (m, 2H), 2.82 (s, 3H), 2.71 (s, 3H).HRMS (ESI): m/z [M + H]+ calc for C24H24N5O5S: 494.1493; found: 494.1492.

9-(4-(Dimethylamino)piperidin-1-yl)-1-(4-methyl-3-nitrophenyl)benzo[h][1 ,6]naphthyridin- 2(1 H)-one (7o - precursor of 23)

Prepared using General Procedure F, using N,N-dimethylpiperidin-4-amine as amine.

Compound 7o was isolated as a yellow solid (215 mg; 77% yield).

¹H NMR (300 MHz, D₆-DMSO): d 8.87 (s, 1 H), 8.23 (m, 2H), 7.85 (d, J = 9.2 Hz, 1 H), 7.77 (d, J = 8.4 Hz, 1 H), 7.70 (dd, J = 8.1 , 2.1 Hz, 1 H), 7.51 (m, 1 H), 6.85 (d, J = 9.4 Hz, 1 H), 6.25 (d, J = 2.6 Hz, 1 H), 3.23 - 3.18 (m, 2H), 2.62 (s, 3H), 2.44 - 2.37 (m, 2H), 2.15 (s, 3H), 2.15 (m, 1 H), 1.66 - 1.62 (m, 2H), 1.28 - 1.23 (m, 2H). HRMS (ESI): m/z [M + H]+ calc for C26H28N5O3: 458.2187; found: 458.2185.

8-(4-(Methylsulfonyl)piperazin-1-yl)-1-(3-nitrophenyl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (7d’n’- precursor of 27)

Prepared using the General Procedure F, reacting intermediate 6d with

1-(methylsulfonyl)piperazine. Compound 7d’n’ was obtained as a brown solid (355 mg; 98%).

HRMS (ESI): m/z [M + H]+ calc for C23H22N5O5S: 480.1336; found: 480.1330. General Procedure G: Nitro to amine reduction with Fe/NH4CI

N-(4-(1-(3-Amino-4-methylphenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin-9- yl)phenyl)methanesulfonamide (8a)

Intermediate 7a (335 g; 0.669 mmol) was suspended in EtOH (40 ml_) and heated to reflux. Fe (224 mg; 4.016 mmol; 6 equiv.) and NH₄CI (215 mg; 4.016 mmol; 6 equiv.) in H₂0 (20 ml_) were added and the mixture heated to reflux. After 2 h, TLC analysis (MeOH:DCM 1 :9) showed that the reaction was completed. The hot mixture was filtered through a Celite pad and the pad further washed with EtOH and MeOH:DCM (2:8). The solvent was evaporated and the crude partitioned between H₂0 and EtOAc. The phases were separated and the aqueous phase was further extracted with EtOAc (3 x). The combined organics were washed with brine, dried over MgS0₄ and taken to dryness to afford the title compound as an off-white solid (330 mg; 100% yield).

N-(4-(1-(3-Amino-5-methylphenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin-9- yl)phenyl)methanesulfonamide (8b)

Prepared using the procedure described for 8a. Compound 8b was obtained as a bright- yellow solid (85 mg; 70% yield).

¹H NMR (300 MHz, D₆-DMSO): 6 9.90 (s, 1 H), 9.07 (s, 1 H), 8.25 (d, J = 9.4 Hz, 1 H), 8.03 (q, J = 8.9 Hz, 2H), 7.70 (s, 1 H), 7.35 - 7.21 (m, 4H), 6.87 (d, J = 9.5 Hz, 1 H), 6.66 (s, 1 H), 6.48 (s, 1 H), 6.29 (s, 1 H), 5.40 (s, 2H), 3.04 (s, 3H), 2.25 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₆H₂₃N₄0₃S: 471.1485; found: 471.1484.

N-(4-(1-(5-Amino-2-methylphenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin-9- yl)phenyl)methanesulfonamide (8c)

Prepared using the procedure described for 8a. Compound 8c was obtained as an orange solid (70 mg; 68% yield).

¹H NMR (300 MHz, D₆-DMSO): 6 9.91 (s, 1 H), 9.1 1 (s, 1 H), 8.30 (d, J = 9.5 Hz, 1 H), 8.12 - 7.97 (m, 2H), 7.53 (d, J = 1.8 Hz, 1 H), 7.32 (d, J = 8.7 Hz, 2H), 7.27 - 7.16 (m, 3H), 6.92 (d, J = 9.4 Hz, 1 H), 6.79 (dd, J = 8.2, 2.3 Hz, 1 H), 6.45 (d, J = 2.3 Hz, 1 H), 5.29 (s, 2H), 3.05 (s, 3H), 1.80 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C26H23N403S: 471.1485; found 471.1485. N-(4-(1-(3-Nitrophenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9- yl)phenyl)methanesulfonamide (8d)

Prepared using the procedure described for 8a. Compound 8d was obtained as a dark yellow solid (60 mg; 92% yield).

¹H NMR (300 MHz, d6-DMSO): d 9.91 (s, 1H), 9.09 (s, 1H), 8.27 (d, J= 9.5 Hz, 1H), 8.11 - 7.95 (m, 2H), 7.60 (d, J = 1.9 Hz, 1 H), 7.36 - 7.27 (m, 3H), 7.23 (d, J = 8.8 Hz, 2H), 6.92 - 6.79 (m, 2H), 6.57 (dd, J= 6.7, 1.2 Hz, 2H), 5.55 (brs, 2H), 3.04 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₅H₂₁N₄O₃S: 457.1329; found: 457.1328.

N-(4-(1-(3-Amino-4-methoxyphenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9- yl)phenyl)methanesulfonamide (8e)

Prepared using the procedure described for 8a. Compound 8e was obtained as an orange solid (85 g; 78% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.93 (s, 1H), 9.07 (s, 1H), 8.26 (d, J= 9.5 Hz, 1H), 8.05 (d, J= 8.7 Hz, 1H), 7.97 (dd, J= 8.7, 1.9 Hz, 1H), 7.37 (d, J= 1.9 Hz, 1H), 7.32-7.18 (m, 4H), 7.05 (d, J= 8.3 Hz, 1H), 6.88 (d, J= 9.4 Hz, 1H), 6.67-6.53 (m, 2H), 5.11 (s, 2H), 3.91 (s, 3H), 3.04 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₆H₂₃N₄O₄S: 487.1435; found:

487.1433.

N-(4-(1-(3-Amino-4-methylphenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-8- yl)phenyl)methanesulfonamide (8a’)

Prepared using the procedure described for 8a. Compound 8a’ was obtained as a bright- yellow solid (80 mg; 71% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.96 (s, 1H), 9.10 (s, 1H), 8.26-8.23 (m, 2H), 7.84 (d, J = 8.7 Hz, 2H), 7.52 (dd, J= 9.4, 2.2 Hz, 1H), 7.30 (d, J= 8.7 Hz, 2H), 7.15 (m, 2H), 6.84 (d, J = 9.4 Hz, 1H), 6.56 (d, J = 2.1 Hz, 1H), 6.48 (dd, J = 7.8, 2.1 Hz, 1H), 5.20 (s, 2H), 3.04 (s, 3H), 2.21 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₆H₂₃N₄O₃S: 471.1485; found:

471.1483. N-(4-(1-(3-Amino-5-methylphenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin-8- yl)phenyl)methanesulfonamide (8b’)

Prepared using the procedure described for 8a. Compound 8b’ was obtained as an orange solid (295 g; 70% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.99 (s, 1 H), 9.09 (s, 1 H), 8.25 - 8.22 (m, 2H), 7.86 - 7.81 (m, 4H), 7.54 (dd, J = 9.4, 2.3 Hz, 1 H), 7.30 (d, J = 8.5 Hz, 2H), 7.21 (d, J = 9.3 Hz, 1 H), 6.84 (d, J = 9.4 Hz, 1 H), 6.61 (s, 1 H), 6.34 (s, 2H), 3.04 (s, 3H), 2.22 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₆H₂₃N₄O₃S: 471.1485; found: 471.1483.

N-(4-(1-(3-Aminophenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin-8- yl)phenyl)methanesulfonamide (8d’)

Prepared using the procedure described for 8a. Compound 8d’ was obtained as a brown solid (140 mg; 85% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.09 (s, 1 H), 8.26 - 8.22 (m, 2H), 7.79 - 7.77 (m, 2H), 7.50 (dd, J = 9.3, 2.2 Hz, 1 H), 7.29 - 7.23 (m, 3H), 7.12 (d, J = 9.3 Hz, 1 H), 6.85 - 6.78 (m, 2H), 6.53 - 6.50 (m, 2H), 5.42 (s, 2H), 2.97 (s, 3H). HRMS (ESI): m/z [M + H]+ calc.

forC₂₅H_2i N₄0₃S: 457.1329; found: 457.1329.

Ethyl 4-(3-amino-4-methylphenylamino)-6-(4-(methylsulfonamido)phenyl)quinoline-3- carboxylate (Intermediate 2 - INT2)

Prepared using the procedure described for 8a. Compound INT2 was obtained as a yellow solid (190 mg; 88%).

¹H NMR (300 MHz, D₆-DMSO): d 10.14 (s, 1 H), 9.88 (s, 1 H), 8.94 (s, 1 H), 8.08 (d, J = 2.0 Hz, 1 H), 8.04 - 7.96 (m, 1 H), 7.92 (d, J = 8.7 Hz, 1 H), 7.67 - 7.50 (m, 2H), 7.39 (d, J = 8.7 Hz, 2H), 7.23 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 7.9 Hz, 1 H), 6.45 (d, J = 2.2 Hz, 1 H), 6.33 (dd, J = 7.9, 2.2 Hz, 1 H), 4.19 (q, J = 7.1 Hz, 2H), 3.02 (s, 3H), 2.1 1 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₆H₂₇N₄O₄S: 491.1753; found: 491.1748.

N-(4-(1-(3-Amino-4-methylphenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin-9- yl)phenyl)butane-1 -sulfonamide (8f - precursor of 15)

Prepared using the procedure described for 8a. Compound 8f was obtained as a bright- yellow solid (163 mg; 91 % yield). ¹H NMR (300 MHz, D₆-DMSO): 69.92 (s, 1H), 9.06 (s, 1H), 8.25 (d, J= 9.5 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1 H), 7.96 (dd, J = 8.7, 1.9 Hz, 1 H), 7.70 - 7.48 (m, 2H), 7.39 (d, J = 1.9 Hz, 1 H), 7.30-7.13 (m, 5H), 6.87 (d, J=9.4 Hz, 1H), 6.61 (d, J = 2.1 Hz, 1H),6.51 (dd, J = 7.8, 2.1 Hz, 1 H), 3.12 (t, J= 7.4 Hz, 2H), 2.25 (s, 3H), 1.76-1.58 (m, 2H), 1.47-1.29 (m, 2H), 0.85 (t, J= 7.3 Hz, 3H). HRMS (ESI): m/z [M + H]+ calc for C29H29N4O3S: 513.1955; found:

513.1954.

N-(3-(1-(3-Amino-4-methylphenyl)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-8- yl)phenyl)methanesulfonamide (8g - precursor of 16)

Prepared using the procedure described for 8a. Compound 8g was obtained as a bright- yellow solid (75 g; 94% yield).

HRMS (ESI): m/z [M + H]+ calc for C26H23N403S: 471.1484; found: 471.1485.

Methyl 4-(1-(3-amino-4-methylphenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin-9- yl)phenylcarbamate (8h - precursor of 17)

Prepared using the procedure described for 8a. Compound 8h was obtained as a bright- yellow solid (147 mg; 100% yield).

¹H NMR (300 MHz, D₆-DMSO): 69.77 (s, 1H), 9.06 (s, 1H), 8.25 (d, J= 9.4 Hz, 1H), 8.08- 7.91 (m, 2H), 7.51 (d, J= 8.3 Hz, 2H), 7.43 (d, J= 1.8 Hz, 1H), 7.22-7.18 (m, 3H), 6.87 (d, J= 9.4 Hz, 1H), 6.61 (d, J= 2.1 Hz, 1H), 6.51 (dd, J= 7.8, 2.1 Hz, 1H), 5.25 (brs, 2H), 3.70 (s, 3H), 2.27 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C27H23N4O3: 451.1765; found: 451.1763.

1-(3-Amino-4-methylphenyl)-9-(pyridin-4-yl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (8i - precursor of 18)

Prepared using the procedure described for 8a. During phase separation, the aqueous phase (pH~5) was adjusted to pH 7 with NaHCC>3. Compound 8i was obtained as an orange solid (75 mg; 100% yield).

¹H NMR (300 MHz, D₆-DMSO): 69.14 (s, 1H), 8.64-8.55 (m, 2H), 8.28 (d, J= 9.5 Hz, 1H), 8.11 (s, 2H), 7.51 (s, 1H), 7.31 -7.23 (m, 2H), 7.20 (d, J= 8.2 Hz, 1H), 6.90 (d, J= 9.4 Hz, 1H), 6.66 (d, J= 2.1 Hz, 1H), 6.49 (dd, J= 7.8, 2.1 Hz, 1H), 5.33 (s, 2H), 2.25 (s, 3H).

HRMS (ESI): m/z [M + H]+ calc for C24H19N4O: 379.1553; found: 379.1552. 5-(1-(3-Amino-4-methylphenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin-9- yl)picolinonitrile (8j - precursor of 19)

Prepared using the procedure described for 8a. Compound 8j was obtained as an orange solid (79 mg; 97% yield).

HRMS (ESI): m/z [M + H]+ calc for C25H18N5O: 404.1506; found: 404.1507.

1-(3-Amino-4-methylphenyl)-9-(6-(trifluoromethyl)pyridin-3-yl)benzo[h][1 ,6]naphthyridin- 2(1 H)-one (8k - precursor of 20)

Prepared using the procedure described for 8a. Compound 8k was obtained as an orange solid (99 g; 100% yield).

¹H NMR (300 MHz, D₆-DMSO): 6 9.15 (s, 1 H), 8.67 (d, J = 2.1 Hz, 1 H), 8.29 (d, J = 9.5 Hz, 1 H), 8.13 (brs, 2H), 7.95 (m, 1 H), 7.88 (m, 1 H), 7.33 (s, 1 H), 7.19 (d, J = 7.8 Hz, 1 H), 6.92 (d, J = 9.4 Hz, 1 H), 6.64 (d, J = 2.1 Hz, 1 H), 6.51 (dd, J = 7.7, 2.1 Hz, 1 H), 5.33 (s, 2H), 2.21 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C25H18F3N4O: 447.1427; found: 447.1426.

1-(3-Amino-4-methylphenyl)-9-(piperidin-1-yl)benzo[h][1 ,6]naphthyridin-2(1 H)-one (8I - precursor of 21)

Prepared using the procedure described for 8a. Compound 8I was obtained as a yellow solid (134 mg; 85% yield).

HRMS (ESI): m/z [M + H]+ calc for C₂₄H₂₅N₄O: 385.2023; found: 385.2025.

1-(3-Amino-4-methylphenyl)-9-morpholinobenzo[h][1 ,6]naphthyridin-2(1 H)-one (8m - precursor of 22)

Prepared using the procedure described for 8a. Compound 8m was obtained as an orange solid (141 mg; 95% yield).

HRMS (ESI): m/z [M + H]+ calc for C23H23N4O2: 387.1816; found: 387.1817. 1-(3-Amino-4-methylphenyl)-9-(4-(methylsulfonyl)piperazin-1-yl)benzo[h][1,6]naphthyridin- 2(1H)-one (8n)

Prepared using the procedure described for 8a. Compound 8n was obtained as a yellow solid (285 mg; 88% yield).

1H NMR (300 MHz, CDCI₃): d 8.79 (s, 1H), 7.94 (dd, J= 16.9, 9.3 Hz, 2H), 7.33 (dd, J= 9.2, 2.6 Hz, 1H), 7.27 (m, 1H), 6.90 (d, J=9.4 Hz, 1H), 6.78 (d, J = 2.6 Hz, 1H), 6.68 (d, J=6.4 Hz, 2H), 3.83 (s, 2H), 3.26 - 3.22 (m, 4H), 2.99 - 2.96 (m, 4H), 2.82 (s, 3H), 2.26 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₄H₂₆N₅0₃S: 464.1751; found: 464.1748.

1-(3-amino-4-methylphenyl)-9-(4-(dimethylamino)piperidin-1-yl)benzo[h][1,6]naphthyridin- 2(1 H)-one (80 - precursor of 23)

Prepared using the procedure described for 8a. The reaction required 12 eq. of Fe and NH4CI and 5 h reaction time at 80°C. During phase separation, the aqueous phase (pH~5) was adjusted to pH 8 with NaHC0₃. Compound 8o was obtained as a yellow solid (85 mg; 46% yield).

¹H NMR (300 MHz, D₆-DMSO): d 8.81 (s, 1H), 8.16 (d, J= 9.4 Hz, 1H), 7.79 (d, J= 9.1 Hz, 1H), 7.46 (dd, J= 9.4, 2.4 Hz, 1H), 7.13 (d, J= 7.8 Hz, 1H), 6.79 (d, J= 9.4, 1H), 6.71 (brs,

1 H), 6.57 (d, J = 1.8 Hz, 1 H), 6.49 - 6.37 (m, 1 H), 5.22 (s, 2H), 3.34 - 3.30 (m, 2H), 2.50 (s, 3H), 2.42-2.36 (m, 2H), 2.16 (s, 6H), 2.16 (m, 1H), 1.70-1.66 (m, 2H), 1.30-1.22 (m, 2H). HRMS (ESI): m/z [M + H]+ calc for C₂₆H₃oN₅0: 428.2445; found: 428.2442.

1-(3-aminophenyl)-8-(4-(methylsulfonyl)piperazin-1-yl)benzo[h][1,6]naphthyridin-2(1H)-one (8d’n’ - precursor of 27)

Prepared using the procedure described for 8a. Compound 8d’n’ was obtained as an orange solid (225 mg; 71% yield).

¹H NMR (300 MHz, CDCI₃): d 8.92 (s, 1H), 8.14 (d, J= 9.4 Hz, 1H), 7.28-7.21 (m, 2H), 7.03 - 6.96 (m, 1 H), 6.86 (d, J = 9.8 Hz, 1 H), 6.77 - 6.68 (m, 2H), 6.46 (brs, 2H), 5.43 (s, 2H), 3.43 (m, 4H,) 3.20 (m, 4H), 2.89 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for

C₂₃H₂₄N₅0₃S: 450.1594; found: 450.1600 General Procedure H: Nitro to Amine reduction with SnC

1-(3-Amino-4-methylphenyl)-9-bromobenzo[h][1 ,6]naphthyridin-2(1 H)-one (Intermediate 3 - INT3)

Intermediate 6a (1.03 g; 2.511 mmol) was suspended in EtOAc (50 ml_) and SnCh (2.86 g; 15.065 mmol; 6 equiv.) was added. The mixture was heated to 85°C and after 2 h, the reaction cooled to r.t. and saturated NaHCC>₃ aq. was added. The phases were separated and the aqueous phase further extracted with ethyl acetate (2 x). The combined organics were washed with brine, dried over MgSCU, taken to dryness to afford INT3 as a brownish solid (650 mg; 68% yield).

¹H NMR (300 MHz, D₆-DMSO): 6 9.1 1 (s, 1 H), 8.25 (d, J = 9.4 Hz, 1 H), 7.91 (d, J = 8.9 Hz, 1 H), 7.78 (dd, J = 8.9, 2.1 Hz, 1 H), 7.18 (d, J = 7.8 Hz, 1 H), 6.99 (d, J = 2.1 Hz, 1 H), 6.90 (d, J = 9.4 Hz, 1 H), 6.53 (d, J = 2.1 Hz, 1 H), 6.45 (dd, J = 7.7, 2.1 Hz, 1 H), 5.24 (s, 2H), 2.22 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for Ci₉Hi₅BrN₃0: 380.0393; found: 380.0390.

General Procedure I: Acylation

N-(2-Methyl-5-(9-(4-(methylsulfonamido)phenyl)-2-oxobenzo[h][1 ,6]naphthyridin-1 (2H)- yl)phenyl)acrylamide (9a; BMX-IN-1)

A stirred solution of 8a (90 mg; 0.191 mmol) in dry THF (20 ml_) was cooled in an ice-bath to -10°C for 20 min. DIPEA (133 uL; 0.765 mmol; 4 equiv.) was added and the mixture stirred for 10 min at T < 4°C. After 10 min, acryloyl chloride was added and the mixture further stirred at -10°C for 10 min. and then 1 h at r.t. THF was then evaporated and the crude redissolved in EtOAc and washed three times with NaHCC>₃ (4%). The organics were dried over MgS0₄ and taken to dryness. The crude was applied in a silica column and eluted with a gradient from 100:0 to 96:4 in DCM:MeOH. The desired fractions were collected and taken to dryness to afford the title compound as a white solid. (20 mg; 20% yield).

¹H NMR (300 MHz, D₆-DMSO): 6 9.85 (s, 1 H), 9.78 (s, 1 H), 9.1 1 (s, 1 H), 8.30 (d, J = 9.4 Hz, 1 H), 8.08 (d, J = 8.7 Hz, 1 H), 7.98 (dd, J = 8.7, 1.9 Hz, 1 H), 7.69 (s, 1 H), 7.52 (d, J = 8.1 Hz, 1 H), 7.23-7.20 (m, 6H), 6.91 (d, J = 9.4 Hz, 1 H), 6.57 (dd, J = 17.2, 10.2 Hz, 1 H), 6.19 (d, J = 17.2 Hz, 1 H), 5.74 (d, J = 10.2 Hz, 1 H), 3.01 (s, 3H), 2.42 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₉H₂₅N₄O₄S: 525.1591 ; found: 525.1586. HPLC Purity: 98.9%. N-(3-Methyl-5-(9-(4-(methylsulfonamido)phenyl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)- yl)phenyl)acrylamide (9b)

Prepared using the procedure described for 9a. Compound 9b was obtained as a light- yellow solid (8 mg; 10% yield).

¹H NMR (300 MHz, D₆-DMSO): d 10.42 (s, 1H), 9.92 (s, 1H), 9.12 (s, 1H), 8.31 (d, J= 9.5 Hz, 1 H), 8.08 (d, J= 8.8 Hz, 1H), 8.00 (dd, J= 8.8, 1.9 Hz, 1H), 7.78 (s, 1H), 7.57 (brs, 1H), 7.44 (d, J= 1.9 Hz, 1H), 7.20 (brs, 4H), 7.09 (s, 1H), 6.92 (d, J= 9.4 Hz, 1H), 6.39 (dd, J = 17.0, 10.0 Hz, 1 H), 6.22 (dd, J= 17.0, 2.2 Hz, 1H), 5.73 (dd, J= 9.9, 2.1 Hz, 1H), 3.03 (s, 3H), 2.36 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₉H₂₅N₄O₄S: 525.1591; found:

525.1576. HPLC Purity: 93.1%.

N-(4-Methyl-3-(9-(4-(methylsulfonamido)phenyl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)- yl)phenyl)acrylamide (9c)

Prepared using the procedure described for 9a. Compound 9c was obtained as a light- yellow solid (40 g; 55% yield).

¹H NMR (300 MHz, D₆-DMSO): d 10.40 (s, 1H), 9.91 (brs, 1H), 9.14 (s, 1H), 8.35 (d, J= 9.5 Hz, 1 H), 8.10 (d, J= 8.7 Hz, 1H), 8.01 (dd, J= 8.7, 1.9 Hz, 1H), 7.83 (dd, J= 8.4, 2.2 Hz, 1H), 7.72 (d, J= 2.1 Hz, 1H), 7.55 (d, J= 8.4 Hz, 1H), 7.32 (d, J= 1.8 Hz, 1H), 7.19 (m, 4H), 6.96 (d, J= 9.4 Hz, 1H), 6.39 (dd, J= 17.0, 10.0 Hz, 1H), 6.22 (dd, J= 17.0, 2.2 Hz, 1H), 5.74 (dd, J= 10.0, 2.2 Hz, 1H), 3.03 (s, 3H), 1.91 (s, 3H). HRMS (ESI): m/z[ M + H]+ calc for C₂₉H₂₅N₄O₄S: 525.1591; found: 525.1587. HPLC Purity: 99.5%.

N-(3-(9-(4-(Methylsulfonamido)phenyl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)- yl)phenyl)acrylamide (9d)

Prepared using the procedure described for 9a. Compound 9d was obtained as a light- yellow solid (27 mg; 50% yield).

¹H NMR (300 MHz, D₆-DMSO): d 10.50 (s, 1H), 9.91 (s, 1H), 9.12 (s, 1H), 8.31 (d, J= 9.4 Hz, 1 H), 8.08 (d, J= 8.7 Hz, 1H), 8.03-7.91 (m, 2H), 7.83 (brs, 1H), 7.63 (t, J= 8.1 Hz,

1H), 7.34 (brs, 1H), 7.18 (m, 5H), 6.92 (d, J= 9.4 Hz, 1H), 6.42 (dd, J= 16.9, 10.0 Hz, 1H), 6.26 (dd, J= 16.9, 2.2 Hz, 1H), 5.77 (dd, J= 9.7, 1.9 Hz, 1H), 3.03 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₈H₂₃N₄O₄S: 511.1435; found: 511.1433. HPLC Purity: 98.0%. N-(2-Methoxy-5-(9-(4-(methylsulfonamido)phenyl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)- yl)phenyl)acrylamide (9e)

Prepared using the procedure described for 9a. Compound 9e was obtained as a light- yellow solid (38 mg; 43% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.73 (s, 1H), 9.10 (s, 1H), 8.29 (d, J= 9.5 Hz, 1H), 8.19- 8.10 (m, 1H), 8.07 (d, J= 8.6 Hz, 1H), 7.97 (d, J= 8.7 Hz, 1H), 7.36-7.20 (m, 8H), 6.91 (d, J= 9.4 Hz, 1H), 6.74 (dd, J= 17.0, 10.2 Hz, 1H), 6.13 (dd, J= 17.0, 2.1 Hz, 1H), 5.68 (dd, J = 10.3, 2.1 Hz, 1H), 4.00 (s, 3H), 3.01 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for

C29H25N4O5S: 541.1540; found 541.1542. HPLC Purity: 97.0%.

N-(2-Methyl-5-(9-(4-(methylsulfonamido)phenyl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)- yl)phenyl)but-2-enamide (10)

Prepared using the procedure described for 9a. Compound 10 was obtained as a pale- yellow solid (30 g; 41%).

¹H NMR (300 MHz, D₆-DMSO): d 9.86 (s, 1H), 9.55 (s, 1H), 9.10 (s, 1H), 8.29 (d, J= 9.5 Hz, 1H), 8.02 (q, J= 8.6 Hz 2H), 7.69 (m, 1H), 7.49 (d, J= 8.1 Hz, 1H), 7.21 (brs, 6H), 6.91 (d, J = 9.4 Hz, 1 H), 6.73 (dd, J= 15.1, 7.2 Hz, 1H), 6.27 (d, J= 15.3 Hz, 1H), 3.02 (s, 3H), 2.41 (s, 3H), 1.83 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C30H27N4O4S: 539.1748; found: 539.1746. HPLC Purity: 99.3%.

3-Methyl-N-(2-methyl-5-(9-(4-(methylsulfonamido)phenyl)-2-oxobenzo[h][1,6]naphthyridin- 1(2H)-yl)phenyl)but-2-enamide (11)

Prepared using the procedure described for 9a. Compound 11 was obtained as a pale- yellow solid (20 mg; 32%).

1 H NMR (300 MHz, d6-DMSO): d 9.39 (s, 1H), 9.10 (s, 1H), 8.29 (d, J = 9.5 Hz, 1H), 8.08- 7.96 (m, 2H), 7.72 (brs, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.16 (m, 6H), 6.91 (d, J = 9.4 Hz, 1H), 6.03 (s, 1H), 2.98 (s, 3H), 2.40 (s, 3H), 2.04 (s, 3H), 1.84 (s, 3H). HRMS (ESI): m/z [M + H]+ calc. for C31H29N4O4S: 553.1904; found: 553.1909. HPLC Purity: 99.3%. N-(5-(9-(4-(Butylsulfonamido)phenyl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-yl)-2- methylphenyl)acrylamide (14)

Prepared using the procedure described for 9a. Compound 14 was obtained as a pale- yellow solid (33 mg; 20%).

¹H NMR (300 MHz, D₆-DMSO): d 9.92 (s, 1H), 9.81 (s, 1H), 9.11 (s, 1H), 8.30 (d, J= 9.4 Hz, 1H), 8.07 (d, J= 8.6 Hz, 1H), 7.98 (d, J= 9.0 Hz, 1H), 7.69 (s, 1H), 7.50 (d, J= 8.1 Hz, 1H), 7.19 (brs, 6H), 6.91 (d, J= 9.5 Hz, 1H), 6.57 (dd, J= 17.1, 9.4 Hz, 1H), 6.19 (d, J= 17.0 Hz, 1H), 5.74 (d, J= 10.1 Hz, 1H), 3.09 (t, J= 7.8 Hz, 2H), 2.40 (s, 3H), 1.74-1.54 (m, 2H), 1.35 (q, J= 7.4 Hz, 2H), 0.83 (t, J= 7.3 Hz, 3H). HRMS (ESI): m/z [M + H]+ calc for C₃₂H₃₁N₄O₄S: 567.2061; found 567.2060. HPLC Purity: 95.2%.

N-(2-Methyl-5-(9-(3-(methylsulfonamido)phenyl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)- yl)phenyl)acrylamide (15)

Prepared using the procedure described for 9a. Compound 15 was purified by semi-prep HPLC with a gradient 25:75 until 50:50 with a mixture (95:5 ACN:NaHCC>₃10 mM) :

(NaHCC>₃10 mM). The title compound was obtained as a white solid (8 mg; 4%).

¹H NMR (300 MHz, CDCIs): d 8.98 (s, 1H), 8.68 (s, 1H), 8.17 (d, J= 8.7 Hz, 1H), 8.07-7.94 (m, 2H), 7.88 (dd, J= 8.7, 1.9 Hz, 1H), 7.54-7.32 (m, 5H), 7.25 (m, 1H), 7.09 (dd, = 8.1, 2.2 Hz, 1H), 6.98-6.84 (m, 2H), 6.56 (dd, J= 16.9, 1.3 Hz, 1H), 6.36 (dd, J= 16.9, 10.2 Hz, 1H), 5.85 (dd, J= 10.2, 1.3 Hz, 1H), 2.98 (s, 3H), 2.34 (s, 3H). HRMS (ESI): m/z [M + H]+ calc. for C₂₉H₂₅N₄O₄S: 525.1591; found 525.1594. HPLC Purity: 94.3%.

Methyl 4-(1-(3-acrylamido-4-methylphenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin-9- yl)phenylcarbamate (16)

Prepared using the procedure described for 9a. Compound 16 was obtained as a pale- yellow solid (55 mg; 36%).

¹H NMR (300 MHz, D₆-DMSO): d 9.75 (s, 2H), 9.09 (s, 1H), 8.29 (d, J= 9.5 Hz, 1H), 8.15- 7.90 (m, 2H), 7.69 (d, J = 2.0 Hz, 1 H), 7.53 - 7.46 (m, 3H), 7.32 - 7.08 (m, 4H), 6.90 (d, J =

9.4 Hz, 1H), 6.58 (dd, J= 17.0, 10.1 Hz, 1H), 6.19 (dd, J= 17.0, 2.0 Hz, 1H), 5.73 (d, J =

10.4 Hz, 1 H), 3.68 (s, 3H), 2.44 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₃₀H₂₅N₄O₄: 505.1870; found 505.1869. HPLC Purity: 97.8%. N-(2-Methyl-5-(2-oxo-9-(pyridin-4-yl)benzo[h][1,6]naphthyridin-1(2H)-yl)phenyl)acrylamide

(17)

Prepared using the procedure described for 9a. Compound 17 was obtained as a yellow solid (29 mg; 20%).

¹H NMR (300 MHz, D₆-DMSO): d 9.76 (s, 1H), 9.17 (s, 1H), 8.56 (s, 2H), 8.32 (d, J= 9.5 Hz, 1H), 8.16-8.12 (m, 2H), 7.73 (s, 1H), 7.52 (d, J= 8.1 Hz, 1H), 7.38 (s, 1H), 7.26-7.20 (m,

3H), 6.94 (d, J= 9.6 Hz, 1H), 6.57 (dd, J= 16.5, 9.6 Hz, 1H), 6.20 (d, J= 16.5 Hz, 1H), 5.74 (d, J= 9.3 Hz, 1 H), 2.43 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₇H₂₁N₄O₂: 433.1659; found: 433.1656. HPLC Purity: 95.8%.

N-(5-(9-(6-Cyanopyridin-3-yl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-yl)-2- methylphenyl)acrylamide (18)

Prepared using the procedure described for 9a. Compound 18 was obtained as a light yellow solid (14 g; 17%).

¹H NMR (300 MHz, D₆-DMSO): d 9.75 (s, 1H), 9.19 (s, 1H), 8.61 (dd, J = 2.3, 0.8 Hz, 1H), 8.32 (d, J= 9.5 Hz, 1H), 8.16-8.13 (m, 2H), 8.06 (dd, J= 8.2, 0.8 Hz, 1H), 7.85 (dd, J= 8.2, 2.3 Hz, 1H), 7.67 (d, J = 2.2 Hz, 1H), 7.51 (d, J=8.1 Hz, 1H), 7.25 - 7.22 (m, 2H), 6.94 (d, J = 9.4 Hz, 1 H), 6.55 (dd, J= 17.0, 10.1 Hz, 1H), 6.17 (dd, J= 17.0, 2.0 Hz, 1H), 5.73 (dd, J = 10.1, 2.0 Hz, 1 H), 2.42 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₈H₂₀N₅O₂: 458.1612; found: 458.1610. HPLC Purity: 95.3%.

N-(2-Methyl-5-(2-oxo-9-(6-(trifluoromethyl)pyridin-3-yl)benzo[h][1,6]naphthyridin-1(2H)- yl)phenyl)acrylamide (19)

Prepared using the procedure described for 9a. Compound 19 was obtained as a yellow solid (20 mg; 18%).

¹H NMR (300 MHz, D₆-DMSO): d 9.77 (s, 1H), 9.18 (s, 1H), 8.66 (s, 1H), 8.32 (d, J= 9.4 Hz, 1H), 8.17-8.14 (m, 2H), 7.87 (s, 2H), 7.67 (d, J = 2.2 Hz, 1H), 7.51 (d, J= 8.0 Hz, 1H), 7.25- 7.22 (m, 2H), 6.94 (d, J= 9.4 Hz, 1H), 6.55 (dd, J= 17.1, 10.2 Hz, 1H), 6.17 (dd, J= 17.1, 2.2 Hz, 1H), 5.73 (d, J= 10.2 Hz, 1H), 2.39 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₈H₂₀F₃N₄O₂: 501.1533; found: 501.1534. HPLC Purity: 98.3%. N-(2-Methyl-5-(2-oxo-9-(piperidin-1-yl)benzo[h][1 ,6]naphthyridin-1 (2H)-yl)phenyl)acrylamide

(20)

Prepared using the procedure described for 9a. Compound 20 was further purified by preparative TLC eluting with DCM:MeOH (96:4) to afford the title compound as a yellow solid (70 mg; 34%).

¹H NMR (300 MHz, CDCI3): d 8.76 (s, 1 H), 8.26 (s, 1 H), 8.03 - 7.79 (m, 3H), 7.38 - 7.27 (m, 2H), 6.97 - 6.90 (m, 2H), 6.62 (d, J = 2.6 Hz, 1 H), 6.33 - 6.26 (m, 2H), 5.67 (t, J = 5.9 Hz,

1 H), 2.74 (m, 4H), 2.29 (s, 3H), 1.48 (m, 6H). HRMS (ESI): m/z [M + H]+ calc for

C27H27N4O2: 439.2129; found: 439.2127. HPLC Purity: 98.2%.

N-(2-Methyl-5-(9-morpholino-2-oxobenzo[h][1 ,6]naphthyridin-1 (2H)-yl)phenyl)acrylamide

(21)

Prepared using the procedure described for 9a. Compound 21 was further purified by preparative TLC eluting with DCM:MeOH (95:5) to afford the title compound as a light-yellow solid (36 mg; 29%).

¹H NMR (300 MHz, CDCIs): d 8.80 (s, 1 H), 8.17 (s, 1 H), 7.97 (t, J = 9.4 Hz, 2H), 7.87 (s, 1 H), 7.38 - 7.28 (m, 2H), 6.99 - 6.92 (m, 2H), 6.64 (d, J = 2.6 Hz, 1 H), 6.41 - 6.26 (m, 2H), 5.70 (m, 1 H), 3.68 (t, J = 4.8 Hz, 4H), 2.88 - 2.62 (m, 4H), 2.28 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₆H₂₅N₄O₃: 441.1921 ; found: 441.1920. HPLC Purity: 97.0%.

N-(2-Methyl-5-(9-(4-(methylsulfonyl)piperazin-1-yl)-2-oxobenzo[h][1 ,6]naphthyridin-1 (2H)- yl)phenyl)acrylamide (22)

Prepared using the procedure described for 9a. Compound 22 was obtained as a yellow solid (45 mg; 25%).

¹H NMR (300 MHz, CDCI₃): d 8.83 (s, 1 H), 8.1 1 - 7.95 (m, 4H), 7.35 - 7.30 (m, 2H), 6.96 (d, J = 9.3 Hz, 2H), 6.68 (d, J = 2.6 Hz, 1 H), 6.34 - 6.30 (m, 2H), 5.71 (m, 1 H), 3.29 - 3.02 (m, 4H), 2.97 - 2.93 (m, 4H), 2.76 (s, 3H), 2.29 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₇H₂₈N₅O₄S: 518.1857; found: 518.1859. HPLC Purity: 96.7%. N-(5-(9-(4-(Dimethylamino)piperidin-1-yl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-yl)-2- methylphenyl)acrylamide (23)

Prepared using the procedure described for 9a. Compound 23 was obtained as a light- yellow solid (9 mg; 10%).

¹H NMR (300 MHz, CDCI₃): d 8.77 (s, 1H), 8.25 (s, 1H), 8.09 - 7.84 (m, 3H), 7.42 - 7.27 (m, 2H), 6.96-6.91 (m, 2H), 6.64 (d, J= 2.6 Hz, 1H), 6.40-6.23 (m, 2H), 5.75-5.58 (m, 1H), 3.30 (t, J= 12.1 Hz, 2H), 2.43-2.29 (m, 3H), 2.29 (s, 3H), 2.26 (s, 6H), 1.70- 1.66 (m, 2H), 1.44 - 1.38 (m, 2H). HRMS (ESI): m/z [M + H]+ calc for C29H32N5O2: 482.2551 ; found: 482.2551. HPLC Purity: 99.5%.

N-(2-Methyl-5-(8-(4-(methylsulfonamido)phenyl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)- yl)phenyl)acrylamide (24)

Prepared using the procedure described for 9a. Compound 24 was obtained as a yellow solid (35 g; 43%).

¹H NMR (300 MHz, D₆-DMSO): d 9.98 (s, 1H), 9.68 (s, 1H), 9.13 (s, 1H), 8.37-8.23 (m,

2H), 7.83 (d, J= 8.6 Hz, 2H), 7.67 (s, 1H), 7.58-7.41 (m, 2H), 7.29 (d, J= 8.7 Hz, 2H), 7.19 (dd, J= 8.0, 2.2 Hz, 1H), 6.98 (d, J= 9.3 Hz, 1H), 6.88 (d, J= 9.4 Hz, 1H), 6.56 (dd, J =

17.1, 10.1 Hz, 1 H), 6.19 (dd, J= 17.0, 2.1 Hz, 1H), 5.74 (d, J= 10.1 Hz, 1H), 3.03 (s, 3H), 2.40 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C29H25N4O4S: 525.1591; found 525.1591. HPLC Purity: 95.1%.

N-(3-Methyl-5-(8-(4-(methylsulfonamido)phenyl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)- yl)phenyl)acrylamide (25)

Prepared using the procedure described for 9a. Compound 25 was further purified by preparative TLC eluting with DCM:MeOH (97:3) to afford the title compound as a yellow solid (10 mg; 9%).

¹H NMR (300 MHz, D₆-DMSO): d 10.39 (s, 1H), 9.98 (brs, 1H), 9.14 (s, 1H), 8.31 -8.28 (m, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.74 (s, 1 H), 7.59 - 7.54 (m, 2H), 7.28 (d, J = 8.3 Hz, 2H), 7.01 -6.96 (m, 2H), 6.89 (d, J= 9.4 Hz, 1H), 6.43 (dd, J= 17.0, 10.0 Hz, 1H), 6.23 (dd, J= 17.0, 2.1 Hz, 1H), 5.76 (dd, J= 9.9, 2.1 Hz, 1H), 3.03 (s, 3H), 2.37 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C29H25N4O4S: 525.1591; found 525.1595. HPLC Purity: 97.1%. N-(3-(8-(4-(Methylsulfonamido)phenyl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)- yl)phenyl)acrylamide (26)

Prepared using the procedure described for 9a. Compound 26 was obtained as a light- yellow solid (20 mg; 18%).

¹H NMR (300 MHz, D₆-DMSO): d 10.42 (s, 1H), 9.94 (brs, 1H), 9.15 (s, 1H), 8.32-8.28 (m, 2H), 7.89 (d, J= 8.2 Hz, 1H), 7.85-7.76 (m, 3H), 7.61 (t, J= 8.1 Hz, 1H), 7.52 (dd, J= 9.3, 2.2 Hz, 1H), 7.29 (d, J= 8.7 Hz, 2H), 7.17 (dd, J= 7.9, 1.1 Hz, 1H), 6.91 (dd, J= 9.4, 8.5 Hz, 2H), 6.43 (dd, J= 17.0, 10.0 Hz, 1H), 6.24 (dd, J= 17.0, 2.1 Hz, 1H), 5.77 (dd, J= 9.9,

2.0 Hz, 1H), 3.03 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C28H23N4O4S: 511.1435; found: 511.1439. HPLC Purity: 98.0%.

N-(3-(8-(4-(methylsulfonyl)piperazin-1-yl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)- yl)phenyl)acrylamide (27)

Prepared using the procedure described for 9a. Compound 27 was obtained as a light- yellow solid (10 g; 6%).

¹H NMR (300 MHz, D₆-DMSO): d 10.44 (s, 1H), 8.97 (s, 1H), 8.19 (d, J= 9.4 Hz, 1H), 7.86 (m, 1H), 7.74 (t, J = 2.0Hz, 1H), 7.59 (t, J=7.9Hz, 1H), 7.30 (d, J = 2.8 Hz, 1H), 7.12 (ddd, J= 7.8, 2.0, 1.0 Hz, 1 H), 6.99 (dd, J= 9.9, 2.9 Hz, 1H), 6.74 (d, J= 9.4 Hz, 1H), 6.64 (d, J = 9.8 Hz, 1H), 6.43 (dd, J= 16.9, 10.0 Hz, 1H), 6.24 (dd, J= 17.0, 2.1 Hz, 1H), 5.78 (dd, J = 10.0 Hz, 2.0 Hz, 1 H), 3.45 (t, J= 5.1 Hz, 4H), 3.19 (t, J= 5.1 Hz, 4H), 2.89 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C26H26N5O4S: 504.1700; found: 504.1703. HPLC Purity: 96.8%.

Ethyl 4-(3-acrylamido-4-methylphenylamino)-6-(4-(methylsulfonamido)phenyl)quinoline-3- carboxylate (28)

Prepared using the procedure described for 9a. Compound 28 was obtained as a yellow solid (10 mg; 10%).

¹H NMR (300 MHz, CDCI₃): d 10.59 (s, 1H), 9.20 (s, 1H), 8.05-7.93 (m, 1H), 7.85-7.75 (m, 3H), 7.46 (s, 1H), 7.23-7.06 (m, 5H), 6.81 (d, J= 7.8 Hz, 1H), 6.47-6.18 (m, 2H), 5.73 (d, J= 9.6 Hz, 1 H), 4.44 (q, J= 7.1 Hz, 2H), 3.01 (s, 3H), 2.33 (s, 3H), 1.45 (t, J= 7.1 Hz, 3H). HRMS (ESI): m/z [M + H]+ calc for C29H29N4O5S: 545.1853; found 545.1849. HPLC Purity: 95.5%. N-(5-(9-Bromo-2-oxobenzo[h][1 ,6]naphthyridin-1 (2H)-yl)-2-methylphenyl)acrylamide (29)

Prepared using the procedure described for 9a. Compound 29 was obtained as an off-white solid (40 mg; 85%).

¹H NMR (300 MHz, D₆-DMSO): d 9.68 (s, 1 H), 9.15 (s, 1 H), 8.29 (d, J = 9.5 Hz, 1 H), 7.94 (d, J = 8.8 Hz, 1 H), 7.79 (dd, J = 8.8, 2.1 Hz, 1 H), 7.66 (s, 1 H), 7.53 (d, J = 8.0 Hz, 1 H), 7.19 (dd, J = 8.0, 2.2 Hz, 1 H), 6.94 (d, J = 9.4 Hz, 1 H), 6.84 (d, J = 2.1 Hz, 1 H), 6.59 (dd, J =

17.0, 10.1 Hz, 1 H), 6.20 (dd, J = 17.0, 2.1 Hz, 1 H), 5.74 (dd, J = 10.0, 2.1 Hz, 1 H), 2.42 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₂₂Hi₇BrN₃0₂: 434.0499; found: 434.0497. HPLC Purity: 92.2%.

General Procedure J: Alkylation

Methyl 4-(2-methyl-5-(9-(4-(methylsulfonamido)phenyl)-2-oxobenzo[h][1 ,6]naphthyridin- 1 (2H)-yl)phenylamino)but-2-enoate (13)

To a stirred solution of 8a (73 mg; 0.155 mmol) and K₂CO₃ (28 mg; 0.202 mmol; 1.3 equiv.) in DMF (2 ml_) at 0°C, methyl-4-bromocrotonate (28 mg; 0.155 mmol; 1 equiv.) in DMF (2 ml_) was slowly added over 1 h, and the mixture stirred at 0°C. The reaction was allowed to warm to r.t. overnight and after 20 h TLC analysis (5% MeOH in DCM) showed complete consumption of starting material. The solvent was evaporated and the crude partitioned between EtOAc and saturated NaHCC aq. The phases were separated and the aqueous phase further extracted with EtOAc (2 x). The combined organics were dried over MgS0₄ and taken to dryness. The crude applied in a silica column and eluted with a gradient from 100:0 to 97:3 (DCM:MeOH). The desired fractions were collected and taken to dryness to afford the title compound as a light-yellow solid (30 mg; 34% yield).

¹H NMR (300 MHz, D₆-DMSO): d 9.09 (s, 1 H), 8.26 (d, J = 9.5 Hz, 1 H), 8.08 - 8.00 (m, 2H), 7.48 - 7.39 (m, 4H), 7.33 (d, J = 8.6 Hz, 1 H), 7.15 (d, J = 7.6 Hz, 1 H), 6.89 (d, J = 9.4 Hz,

1 H), 6.79 - 6.72 (m, 1 H), 6.66 (d, J = 2.0 Hz, 1 H), 6.48 (dd, J = 7.7, 2.0 Hz, 1 H), 6.05 (d, J = 15.7 Hz, 1 H), 5.38 (m, 0.5H), 5.28 (brs, 2H), 4.57 (d, J = 4.5 Hz, 1 H), 4.42 (dt, J = 14.3, 7.3 Hz, 0.5H), 3.65 (s, 3H), 3.10 (s, 3H), 2.21 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C₃₁ H₂₉N₄O₅S: 569.1853; found: 569.1853. HPLC Purity: 99.5% (2 isomers with 1 :5.3 ratio).

N-(4-(1-(3-(3-Cyanoallylamino)-4-methylphenyl)-2-oxo-1 ,2-dihydrobenzo[h][1 ,6]naphthyridin- 9-yl)phenyl)methanesulfonamide (12)

Prepared using the procedure described for 13. Compound 12 was obtained as a pale- yellow solid (25 mg; 39%). ¹H NMR (300 MHz, D₆-DMSO): d 9.10 (s, 1 H), 8.27 (d, J = 9.4 Hz, 1 H), 8.08 - 8.03 (m, 2H), 7.47 - 7.35 (m, 4H), 7.33 (d, J = 8.5 Hz, 1 H), 7.17 (d, J = 7.8 Hz, 2H), 6.89 (dd, J = 9.4, 2.3 Hz, 2H), 6.67 (m, 1 H), 6.49 (dd, J = 7.8, 2.3 Hz, 1 H), 5.87 (dd, J = 31.2, 13.6 Hz, 0.5H), 5.30 (brs, 2H), 4.64 (d, J = 6.6 Hz, 0.5H), 4.54 (d, J = 5.0 Hz, 0.5H), 4.26 (dt, J = 13.5, 6.5 Hz, 0.5H), 3.11 (s, 3H), 2.20 (s, 3H). HRMS (ESI): m/z [M + H]+ calc for C31H25N5O3S:

536.1751 ; found: 536.1751. HPLC Purity: 99.0% (2 isomers with 1 :1.65 ratio).

Solubility, Lipophilicity, and PAM PA Permeability

The modifications introduced in the scaffold also sought to improve the physicochemical profile of the new analogues. BMX-IN-1 is a lipophilic molecule (cLogP = 3.94) with limited water solubility (LogS = -5.98) (see Table 1 below). 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. Most interestingly, installation of 1-(methylsulfonyl)piperazine at the 7-position gave compound 27, the analogue with the best in silico lipophilicity and water solubility profile: cLogP = 2.32 and LogS = - 4.36.

Considering that BMX-IN-1 does not have an optimal physicochemical profile, it was anticipated that 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. For assessment of drug permeability, we relied on parallel artificial membrane permeability assay (PAMPA) performed at Pion Inc. The PAMPA Evolution™ instrument was used to determine permeability and we observed that the vast majority of the analogues have a high permeability (Table 1).

Once again, the solubilizing motifs introduced in the sulfonamide region led to an increase in effective permeability. Based on the values of cLogP calculated for molecules 9-29, it is believed that 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.

From the data given in Table 2, it can be seen that the analogues with higher cLogP (14, 16, 19, 28) are the compounds with more limited solubility (LogS), which was also observed in the PAMPA assay. In addition, very lipophilic compounds such as 11 and 13 display the highest permeability rates but also the least lipophilic compounds (21 , 22, 26 and 27) display good permeability rates, therefore reinforcing the hypothesis In summary, the modifications introduced did improve the overall profile of the analogues in most cases, including compounds 26 and 27.

Colloidal Aggregation

Colloidal aggregation is the major source of false positive readouts in screening assays. To rule out unspecific binding of compounds 9-29 to BMX particle sizes were measured using dynamic light scattering (DLS). The data shows that, albeit with limited solubility, the synthesized compounds do not form aggregates at the relevant inhibitory concentrations (see Table 1).

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.

Artificial Membrane Permeability (PAMPA)

The PAMPA Evolution™ instrument was used to determine permeability, at Pion Inc. In PAMPA, 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 10⁶ 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.

In silico cLog and LogS cLogP and LogS were calculated using SwissADME software (Daina et ai). 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 10⁶ 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 IC₅₀ concentration (green - no aggregation at IC₅₀ concentration; red - aggregation at IC₅₀ concentration.

Structure-Activity Relationship

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.

In order to explore the SAR, the library of compounds was tested in an IC₅₀ enzymatic assay against recombinant BMX, performed by Eurofins-CEREP (France). The results are shown in Figure 1.

We initially focused our modifications in two main regions corresponding to -D and -R⁶ in the compounds of formula (II), and also introduced minor changes region corresponding to -A- in the compounds of the invention, for systematic modulation. Covalent inhibition of BMX is thought to occur via alkylation of Cys496, which is located at the lip of the ATP binding site. In a stepwise approach, the importance and tolerability of different substituents in the regions corresponding to -D and -A- were investigated, which regulates the electrophilic attack by the cysteine and other potential nucleophiles.

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.

Moving the methyl substituent to the phenylene 6-position abolishes target inhibition (9C) while a methyl positioning in the 5-position slightly improved it by 2-fold (9B). Even more striking is the effect of no substituent in the ring (9D) increasing inhibition by 6-fold. Since the electronic influence of the methyl substituents in the different positions is not expected to account for these differences, it is believed that a conformational effect may play an important role. A 6-substituent may increase the constraints for fitting into the pocket, while the removal of the methyl groups affords less spatial restriction.

The binding process is partially regulated by the nucleophilic attack at the acceptor group,

-M. As such, the modification of the acceptor group was envisioned to determining if different electrophiles would influence binding.

The results clearly show that modifications of the acceptor group can lead to abrogation of binding. Introducing one (10) or two (11) terminal methyl groups to an alkene acceptor decreases inhibition. Also, inverting the acceptor moiety (13), or introducing a conjugated alkene and nitrile (12), can limit binding. Therefore, the acceptor group was not modified in subsequent scaffolds.

The focus of the research then moved on to the nature of the groups at the -R⁶ position. Docking studies with BMX-IN-1 had previously suggested that a major interaction occurs at the distal sulfonamide which regulates the interaction with Lys445 (Liu et al. ACS Chem. Biol.). To evaluate the importance of this region in the activity of the molecule, we replaced the phenyl-containing group at -R⁶ of BMX-IN-1 with a variety of alternative aromatic and non-aromatic substituents.

Compound 14 was prepared in order to understand if this position could be a good option for placing a long chain substituent. Clearly, the results showed that a long substituent is not tolerated most probably because fitting into the pocket is impaired. Based on this information, we explored new substituents that could afford different interactions. Moving the sulfonamide to the meta position of the phenyl ring at the 6-position (15) only afforded a 2-fold gain in potency.

To gather further insight into the relevance of the interaction with Lys445 new modalities were provided at the quinolone 6-position. 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!.).

Following the same principles, the use of 4-pyridine (17) and substituted 3-pyridines (18 and 19) sought to lower lipophilicity by replacing the aryl ring with a heterocycle.

Unfortunately, neither the carbamate (16) nor 4-pyridine (17) substituents provided large increase in activity (only a 1-fold increase) and the substituted pyridines (18 and 19) decreased affinity by 3- and 9-fold.

One of the research goals was to improve the drug likeness of the compounds for use. With an eye towards optimization of physicochemical properties it was an aim of the project to lower the lipophilic profile of BMX-IN-1 and to improve its limited solubility. Here, consideration was given to disruption of possible tt-p stacking interactions due to the high number of aromatic rings and introduced reduced forms of parent heterocycles, such as piperidines and piperazines, aiming to improve the drug profile.

It was observed that the analogues bearing piperidine (20), dimethylamino-piperidine (23) and morpholine (21) afforded similar inhibition while the sulfonamide-piperazine (22) performed slightly better improving activity by 3-fold. These data suggested that a major interaction was not occurring at this protein site and in order to confirm these observations compound 29 was prepared.

By virtually removing any group capable of interacting with the Lys445 residue, the binding was not affected. This observation prompted us to a more thorough assessment of alternative regions of the parent molecule. More specifically, the relevance of the tricyclic core was assessed by disrupting the central unit. The acceptor group and the sulfonamide regions were not modified but the fused pyridinone ring was cleaved leaving a free ester functionality (28). The results clearly show that the tricyclic central core is required since this analogue lost 7-fold in potency. With this information in hand, we sought to prepare compound 24. Since the initial analogues with substituents at the quinoline 6-position only improved binding slightly, compounds having substituents at the 7-position were considered as alternatives.

A striking improvement of 14-fold in potency clearly showed that the 7-position is the best positioning for substituents to the quinolone-containing core. Analogues 25, 26 and 27 were also prepared, in which the structural groups from the 6-substituted analogues were used, to afford compounds having a preferred overall profile. From the earlier work on the

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.

All the new analogues displayed improved potency in comparison to BMX-IN-1 (14-, 6- and 4-fold, respectively for compounds 25, 26 and 27). Importantly, in order to obtain a direct comparison between leads 24-27, all the compounds were tested in the same assay, once again using BMX-IN-1 as a control in the experiment and re-testing compound 24. These results demonstrate that positioning substituents at the quinoline 7-position opens new possibilities regarding effective binding in the pocket and potential conjugation without affecting activity.

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. Given the structural similarity between 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. On the other hand, the LE improvement is driven by the increased potency of all analogues rather than a decrease in the size of the molecule.

To date, all the reported BMX inhibitors also display the ability to inhibit Bruton’s tyrosine kinase (BTK). In order to determine if our leads were selective binders of BMX, we also evaluated binding against BTK (Eurofins). For the BTK IC₅₀ assay analogues with higher BMX inhibitory capacity - 24 and 25 - were selected, as well as analogue 27, which presents the best LE and LipE improvement (and also offers the possibility of derivatization).

The results showed that all the compounds are potent BTK inhibitors, in the low nanomolar range (Table 2 below). The same inhibitory trend is observed with an increase of 62-, 33- and 15-fold potency with 25, 24 and 27, respectively, in comparison to BMX-IN-1.

Interestingly, 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 BTK BMX BTK

C50 (fiM) IC50 (nM) (LE/LipE) (LE/LipE)

50.2 362 0.26/3.36 0.23/2.50

7.5 11.1 0.29/4.17 0.29/3.99

3.5 5.8 0.30/4.43 0.30/4.22 9.1 Nd 0.30/4.45 Nd

13.7 24.3 0.30/5.54 0.29/5.29

LE— Ligand efficiency; LipE - Lipophilic efficiency; Nd - not determined

Biochemical Kinase Assay

BMX kinase activity (IC50) was performed at CEREP-France. Briefly, the inhibition of the human recombinant Bmx kinase is quantified by measuring the phosphorylation of the substrate ^oΐ^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.

The results are shown in Figure 1.

Differential Scanning Fluorimetry

DSF, a fluorescence-based thermal shift assay, was used to study HiS₆-BMX thermal stabilization upon ligand binding (Niesen et ai.\ Fedorov et ai), thus providing an indication of the directed interactions between the target-protein and the reported inhibitors.

The purified recombinant human HiS₆-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.

As shown in Table 3, 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.

Table 3: Melting temperature (T_m) shift calculated with a DSF assay

DSF was performed in MicroAmp™ EnduraPlate™ Optical 96-Well Clear Reaction Plates with Barcode (Applied Biosystems, Life Technologies, California, USA) using a

GuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Pre-incubation of the protein with the compound for 2 hours at 4°C was required prior to DSF experiments. The final reaction mixture (20 pL of total volume) contained 4 pg of His6-BMX, 4-fold of Protein Thermal Shift™ Dye (Applied Biosystems) diluted in protein buffer solution, and 100 pM of compound. The temperature was increased from 25°C to 90°C with an increment rate of 0.016°C/s. Excitation and emissions filters were applied for Protein Thermal Shift™ Dye (470 nm and 520 nm, respectively) and for ROX reference dye (580 nm and 623 nm, respectively). The melting temperatures were obtained by taking the midpoint of each transition.

Surface Plasmon Resonance

The putative interaction of the synthetic compounds with BMX in real time was analysed by using Surface Plasmon Resonance (SPR). SPR is a sensitive spectroscopic method that can be used as a primary tool to screen interacting molecules or as a validation tool for interactions previously identified by other methods () [55]

To validate the HTRF and DSF results reported here, compounds that showed a significant potency gain or loss (fold) against control BMX-IN-1 were injected at different concentrations on BMX immobilized surfaces (sensorgrams not shown). As expected, BMX-IN-1 was shown to interact with BMX with high affinity ( KD = 69 nM) as observed in the biochemical assay. The results show that compounds 9C, 10 and 12 interact transiently with the target, with fast association and dissociation kinetics, making it possible to calculate only the affinity at steady state ( K_Dss ) This observation is in accordance with the lack of inhibitory capacity observed in the enzymatic assay and it reinforces the hypothesis that steric hindrance is hampering the protein-compound association The enzymatic assay results are also supported by the kinetic evaluation of 9D and 9E interaction with BMX. One compound, 9D, shows a KD value in the 1-digit nanomolar range whilst compound 9E has lower affinity than the control BMX-IN-1 (69 nM).

Higher affinity interactions were observed, as expected, for compounds 24 to 27, showing comparable association rates (K_on from 5.4 x 10⁴ M ¹S ¹ to 1. 4 x 10⁵ IV s^-1) but, most importantly, very slow dissociation rates (K_0ff < 1 x 10^-4 s^-1), in agreement with the covalent nature of the interaction.

Table 4: Kinetic Constants Calculates from Surface Plasmon Resonance

Nd - not determined;‘Immeasurable KD due to very prolonged off-rates (outside instrument specification).

SPR Experiments were carried out in a Biacore 4000 instrument (Biacore AB, GE

Healthcare Life Sciences, Uppsala, Sweden) at 25°C. 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

400 mM 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide and 100 mM N-hydroxysuccinimide for 10 min. HBS-N (10 mM HEPES pH 7.4, 150 mM NaCI) was used as the background buffer. 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.

Compounds were pre-diluted in DMSO to 50 times the desired highest tested concentration and diluted afterwards in running buffer (20 mM HEPES pH 7.4, 150 mM NaCI, 1 mM DTT, 0.1 mM EGTA, 0.05 % (v/v) TWEEN-20, 5 mM MgCI₂) in order to reach 2% of DMSO concentration. A DMSO solvent correction (1 %-3%) was performed to account for variations in bulk signal and to achieve high-quality data.

Each compound was injected over immobilized HiS₆-BMX for 220 s (30 pl_ min^-1 ; association phase) followed by 600 to 2,000 s of buffer flow (dissociation phase) at a maximum concentration of 0.5 mM or 10 mM for high and low affinity binders, respectively, and diluted five times in 2-fold dilution series. All sensorgrams were processed by first subtracting the binding response recorded from the control surface (reference spot), followed by subtracting the buffer blank injection from the reaction spot. All datasets were fit to a simple 1 : 1

Langmuir interaction model with the provided Biacore 4000 Evaluation software, to determine kinetic rate constants (k_on, k_0ff) or steady-state affinity (KD_ss).

Native Mass Spectrometric Analysis

In order to further confirm the covalent binding mode between the protein and the ligand(s), a mass spectrometry study was performed using compound 24 as probe. The truncated human BMX was analysed by native MS and the protein mass found was 30899 Da (see Figure 2(A)).

The protein was then treated with 24 and directly analysed by denaturating MS (as detailed further below). The mass found upon incubation with 24 is 31 ,424 Da which is 525 Da larger than the apo-form of hBMX (Figure 2(B)). This result suggests covalent conjugation of a single molecule of 24 to hBMX. Furthermore, proteomics analysis of drug conjugated hBMX indicates the drug covalently interacts with the cysteine residue at position 496 (Figure 2(C)).

For native MS analysis, 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%

H20 and 0.1% formic acid) at a flow rate of 10 pL/min for 10 min, eluted and separated onto a C18 column (Acclaim PepMap100, C18, 75 pm x 15 cm, Thermo Scientific) with a linear gradient from 0% to 100% buffer B (50% isopropanol, 45% acetonitrile, 5% H O and

0.1 % formic acid) at a flow rate of 300 nL/min in 50 min.

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. For full MS scan, the mass range was set to 335 to 2,000 m/z at a resolution of 60,000. For tandem MS scan, the CID normalized energy was 35%.

Crystallization and Structure Determination of BMX in Complex with Inhibitor

To further characterize the inhibition mechanism and binding mode of the compounds described herein, a variety of commercial crystallization screens were tested in order to obtain a protein crystal suitable for X-ray diffraction.

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. Not surprisingly, 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)). Contrary to what was proposed by the docking studies with BMX-IN-1 , 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

(Figure 3(C)), where the Asp554 side chain is positioned in the back cleft, away from the ATP binding pocket, and the Phe555 aromatic ring points up into the gatekeeper region blocking the b 3 Lys445-aC Glu460 ion pair formation. Both the activation loop and the DFG- out-like conformation are similar to what is observed in the only reported BMX crystal structure with the non-covalent inhibitors Dasatinib and PP2 (Muckelbauer et a!.). The positioning of the 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 positioning of the sulfonamide-substituted phenyl ring is also of outmost importance. Contrary to the docking results with BMX-IN-1 , this group is not interacting with any important residue and it is in fact pointing out of the ATP pocket (see Figure 3(D)). This observation is of critical importance because it allows for the introduction of a linker or chemical handle in this region of the molecule. Since it is not sterically hindered by other residues, one can expect the linker or handle to remain outside the pocket, and therefore not to significantly influence the inhibitor binding capacity.

The guidelines for plasmid construction and vector cloning are described by

Muckelbauer et al. The expression of BMX protein using Sf-9 cells, as well as the purification process, were optimized to improve sample quality at the end of purification, in order to increase the likelihood of protein crystallization (manuscript in preparation). The purified BMX tyrosine kinase was concentrated to a final concentration of 10 mg/mL

(according to Muckelbauer et al.) and pre-incubated for 2 hours at 20°C with a 2-fold concentration of the inhibitor 24. The trials were carried out using the sitting-drop vapor diffusion method on the Mosquito® LCP crystallization robot (TP Labtech Ltd, Hertfordshire, UK). The drops consisted of 0.150 pL of the reservoir solution mixed with an equal volume of the protein sample, equilibrated against a 45 pL reservoir. The crystals appeared after 2 days in a lead condition consisting of 0.2 M imidazole-malate buffer, pH 5.5, with 42% v/v PEG 600.

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.\

Potterton et at.) using the PDB entry 3SXS (Muckelbauer et at.) as a search model, without including ligands and water molecules.

Two independent copies of the search model were located in the crystal structure, and model rebuilding was carried out with BUCANEER (Potterton et al.) and COOT (Cowtan). Initial structure refinement was undertaken with REFMAC (Emsley et a!.). The

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|Fo|-|Fc| and |Fo|-|Fc| electron density maps. In the final refinement cycles, hydrogen atoms were added and refined in calculated positions, Translation-Libration-Screw rigid-body anisotropic atomic displacement parameters were refined, water molecules added automatically and the relative weights between the crystallographic and stereochemical energy terms optimized.

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/.).

Kinase Selectivity

As mentioned previously, most of the BMX inhibitors reported to date offer poor selectivity since they are both BMX and BTK inhibitors. Their cellular effect is often attributed to off- target activity either upstream or downstream of BMX signalling pathways. In order to investigate in which targets our new analogues could have an effect, we tested the most potent analogue (25) against a panel of 36 BMX-related kinases in the Eurofins DiscoveRx’s KINOMEscan platform at the concentration of 1 mM.

From the wide number of accessible cysteines residues distributed across the whole kinome not all are available for covalent modification [11-13] 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. Also included were kinases involved in upstream (Src, FAK, PI3K, mTOR, PDK1) and downstream (Akt, PAK1 , TAM) 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 results for primary screen binding interactions at 1 mM concentration are reported as %

DMSO control.

As stated above, 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. More specifically, 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. These results show that compound 25 is a good probe molecule for TEC kinases and suggest that any cellular activity mediated by 25 is probably due to inhibition of any of the TEC kinases rather than any off-target inhibition of upstream and downstream BMX regulators.

Cellular Activity Assay

LNCaP and PC-3 Cell Growth Assay

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).

To determine the effect of the most potent analogues in prostate cancer cells, the ability of compounds 24-27 to inhibit the proliferation of LNCaP and PC-3 prostate cancer cell Lines was tested, using CellTiter-Glow®.

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). In LNCaP - androgen- receptor positive cells a different profile was observed. 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.

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

compounds (2-fold) in 100 mI_ media were added to the cells 24 hours later. After 96 hours incubation cellular viability was assessed by CellTiter-Glo (Promega) according to the manufacturer’s instructions. The values were normalized to vehicle and IC50 was calculated using GraphPad Prism software.

Propidium Iodide assay

In order to determine whether the growth inhibition was due to apoptosis, we carried out Fluorescence assisted cell sorting (FACS) analysis, using Propidium Iodide staining (PI). LNCaP cells were incubated with BMX-IN-1 and 24-27 for 64h, at 10 mM and results showed that no marked differences were observed in the percentage of necrotic events when compared to the vehicle control, showing that in these conditions, these analogues do not cause increased cell death (see Figure 4).

It is not surprising that all the compounds show a low proliferation inhibitory potential in prostate cancer cell lines and it remains questionable whether modulation of BMX alone is relevant or not towards anti-proliferative effects (Price et at). In fact, a large body of evidence is present in the literature showing that selective or dual BMX/BTK inhibitors have poor anti-proliferative effects in BMX-dependent models, most probably from dynamic compensation of signalling mechanisms. Focus has been made on the modulation of BMX activity to sensitize cells to others therapeutic agents since anti-proliferative effects are only observed in combination with inhibitors of related pathways (Fox et ai\ Fox et a!.).

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). Available data shows that BMX is a key regulatory protein but not an effector and the combinatorial approach is believed to be the most effective.

Consequently, this collection of compounds can become useful molecules for combinatorial treatments.

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,

USA) into round-bottom FACs tubes, and washed with 10 % FBS in PBS. Cells were then re-suspended in 5 pg/mL of propidium iodide diluted in wash buffer and analyzed directly after 15 minutes using an LSR Fortessa X-20 flow cytometer equipped with a 488 nm laser and a 670LP and 695/40BP combination of filters. The results for compounds 24-27 and BMX-IN-1 are shown in Figure 4 as percentages of controls (i.e. vehicles) and represent average ± SD (of triplicates).

Additional Data

Irreversible binding efficacy relative to BMX-IN-1

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.

Therefore, we evaluated the irreversible binding efficiency of our rationally designed compounds. The kinetic analysis is presented in Table 7, reveals that compound 25 exhibits the best binding fit with the target, with a binding affinity of 323 pM. This represents an increase in excess of 10-fold relative to BMX-IN-1 (Kl: 4.07 nM). The other leads, display a similar binding affinity among themselves (1.93-2.52 nM), lower than 25 and approximately 2-fold higher than BMX-IN-1. However, the rate of covalent bond formation of the bound inhibitor (determined by kKmact) shows that compounds 24, 25, and 26 showed slightly improved efficiency (0.335, 0.378 and 0.443 min^-1, respectively) in comparison to BMX-IN-1 (0.217 min^-1) and 27 (0.166 min^-1). Consequently, the irreversible binding efficiency of 25 (19.4 pM^-1s^-1) is the highest of the series, whereas BMX-IN-1 shows the lowest result (0.89 pM^-1s^-1) relative to the remaining inhibitors. Overall, these results provide quantitative evidence that the improved activity is mostly driven by changes in the binding

complementarity between the compound and target rather than faster rate of covalent binding. Thus, taking into account that all the analogues have the same Michael acceptor moiety, the enhanced activity must be a result of the structural modifications introduced in the scaffold.

Table 7. Determination of the kinetic parameters Ki, k_inact, k_inact/K|.^a

^aResults tested in duplicate, showing mean ± S.D..

bResults obtained from two independent studies, showing mean ± S.D..

cValue with a 0.06 pM^-1s^-1 deviation from published results. (Wang et ai). Intracellular BMX inhibition

To validate target affinity and identification for 25, we performed an intracellular target engagement kinase assay with HEK293 cells expressing NanoLuc^®-BMX fusion vector with Promega’s NanoBRET^™ TE Intracellular Kinase Assay. The cell proliferation depends on BMX kinase activity that was used to monitor the cellular activity of the compounds (IC50). As shown in Table 8, the IC50 determination showed the inhibitory capacity of 25 (IC50: 44.8 nM) is 10 times greater than BMX-IN-1 (IC50: 495 nM), which aligns with the previous

observations of an increased biochemical potency with similar activity difference.

Assay performed at Reaction Biology Corporation (USA), with concentrations tested in duplicate, showing mean ± S.D. Cells were treated for 1 h and IC50 values were calculated and plotted by using GraphPad Prism 8 based on a sigmoidal dose response curve.

Table 8: Intracellular target engagement in HEK293 cells transiently transfected with BMX expressing NanoLuc^®-BMX.

ln-cell target engagement was performed at the Reaction Biology Corporation (USA) using the NanoBRET™ technology. Very briefly, 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).

Cancer cell growth inhibition by BMX inhibitors.

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. In a previous experiment (unpublished results) 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. The study included 24, BMX-IN-1 and the structurally similar compounds 10 and 11 which do not bind to BMX (Figure 1).

The results presented in Table 9 show that 10 and 11 (non-binders) have little or no effect on viable cell growth of the majority of the tested cell lines. 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). In contrast, androgen receptor negative cells (DU145 and PC3) were overall more resistant to treatment. In addition, 24 showed potent inhibitory effects against LNCaP and 22RV1 but also against PC3, which are androgen receptor negative cells. Furthermore, both compounds were also potent inhibitors of viable cell growth for RS4 (11) (lymphoblast) and DAUDI (T-lymphoblast) cells, in which BTK is highly overexpressed. Altogether, these results demonstrate BMX inhibition impacts viable cell growth of prostate cancer cells and prompted us to further investigate the importance of the androgen receptor and related BMX pathways in these cell lines.

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.

Co-treatment of LNCaP cells with 24-26 and androgen receptor antagonist, PI3K and AKT inhibitors.

As shown above, BMX inhibition alone induces limited cell death in BMX-expressed cell lines owing to the existence of compensatory mechanisms in signalling pathways. To evaluate the use of 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. For this purpose, 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. An optimization study was performed by screening several concentrations to determine the ideal conditions to obtain initial viability above 80% with the individual inhibitors alone. Based on these results, we tested 24 (at 3 mM), 25 (5 pM) and 26 (6 pM) with AKT1/2 (1 pM), Flutamide (50 pM) and LY294002 (3 pM).

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.

Although the control concentrations of 24-26 and the inhibitors did not have an effect on reducing cell viability upon co-treatment, a marked viability decrease was observed in all tested conditions. With AKT1/2 a decrease in cell viability ranging from 48% (with 24) to 63% (with 26) was observed, relative to control AKT1/2. Wth Flutamide, the most effective combination was with compound 25 (63% cell viability reduction) and the least effective with 24 (44% reduction). Finally, co-treatment with LY294002 decreased cell viability by 35%

(with 24 and 26) and 59% (with 25). Overall, these results demonstrate a synergistic effect between 24-26 and AKT1/2, Flutamide and LY294002 in cancer cell proliferation capable of overcoming the compensatory mechanisms of BMX inhibition, and open the possibility of becoming useful molecules for drug combination approaches. Targeted cell cytotoxicity in patient samples

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.

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. As can be seen in Figure 6, compound 25 has an“on target” effect in 7 out of 1 1 patient samples.

Sequence Listings

SEQ ID No. 1: BMX Amino Acid Sequence

MDTKSILEELLLKRSQQKKKMSPNNYKERLFVLTKTNLSYYEYDKMKRGSRKGSIEIKKI RCVEKVNLEEQTPVERQYPFQIVYKDGLLYVYASNEESRSQWLKALQKEIRGNPHLLVKY HSGFFVDGKFLCCQQSCKAAPGCTLWEAYANLHTAVNEEKHRVPTFPDRVLKIPRAVPVL KMDAPSSSTTLAQYDNESKKNYGSQPPSSSTSLAQYDSNSKKIYGSQPNFNMQYIPREDF PDWWQVRKLKSSSSSEDVASSNQKERNVNHTTSKISWEFPESSSSEEEENLDDYDWFAGN ISRSQSEQLLRQKGKEGAFMVRNSSQVGMYTVSLFSKAVNDKKGTVKHYHVHTNAENKLY LAENYCFDSIPKLIHYHQHNSAGMITRLRHPVSTKANKVPDSVSLGNGIWELKREEITLL KELGSGQFGVVQLGKWKGQYDVAVKMIKEGSMSEDEFFQEAQTMMKLSHPKLVKFYGVCS KEYPIYIVTEYISNGCLLNYLRSHGKGLEPSQLLEMCYDVCEGMAFLESHQFIHRDLAAR NCLVDRDLCVKVSDFGMTRYVLDDQYVSSVGTKFPVKWSAPEVFHYFKYSSKSDVWAFGI LMWEVFSLGKQPYDLYDNSQWLKVSQGHRLYRPHLASDTIYQIMYSCWHELPEKRPTFQ QLLSSIEPLREKDKH

>sp|P51813|BMX_HUMAN Cytoplasmic tyrosine-protein kinase BMX OS=Homo sapiens OX=9606 GN=BMX PE=1 SV=1

Reference: Uniprot (https://vwvw.uniprot.orq/uniprot/P51813) SEQ ID No. 2: BTK sequence - Isoform BTK- A: canonical sequence

MAAVILESIFLKRSQQKKKTSPLNFKKRLFLLTVHKLSYYEYDFERGRRGSKKGSIDVEK ITCVETWPEKNPPPERQIPRRGEESSEMEQISIIERFPYPFQVVYDEGPLYVFSPTEEL RKRWIHQLKNVIRYNSDLVQKYHPCFWIDGQYLCCSQTAKNAMGCQILENRNGSLKPGSS HRKTKKPLPPTPEEDQILKKPLPPEPAAAPVSTSELKKWALYDYMPMNANDLQLRKGDE YFILEESNLPWWRARDKNGQEGYIPSNYVTEAEDSIEMYEWYSKHMTRSQAEQLLKQEGK EGGFIVRDSSKAGKYTVSVFAKSTGDPQGVIRHYWCSTPQSQYYLAEKHLFSTIPELIN YHQHNSAGLISRLKYPVSQQNKNAPSTAGLGYGSWEIDPKDLTFLKELGTGQFGWKYGK WRGQYDVAIKMIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANG CLLNYLREMRHRFQTQQLLEMCKDVCEAMEYLESKQFLHRDLAARNCLVNDQGVVKVSDF GLSRYVLDDEYTSSVGSKFPVRWSPPEVLMYSKFSSKSDIWAFGVLMWEIYSLGKMPYER FTNSETAEHIAQGLRLYRPHLASEKVYTIMYSCWHEKADERPTFKILLSNILDVMDEES

>sp|Q06187|BTK_HUMAN Tyrosine-protein kinase BTK OS=Homo sapiens OX=9606 GN=BTK PE=1 SV=3

Reference: Uniprot (https://www.uniprot.Org/uniprot/Q06187#sequences)

SEQ ID No. 3: BTK sequence - Isoform BTK-C

MASWSIQQMVIGCPLCGRHCSGGEHTGELQKEEAMAAVILESIFLKRSQQKKKTSPLNFK KRLFLLTVHKLSYYEYDFERGRRGSKKGSIDVEKITCVETWPEKNPPPERQIPRRGEES SEMEQISIIERFPYPFQVVYDEGPLYVFSPTEELRKRWIHQLKNVTRYNSDLVQKYHPCF WIDGQYLCCSQTAKNAMGCQILENRNGSLKPGSSHRKTKKPLPPTPEEDQILKKPLPPEP AAAPVSTSELKKWALYDYMPMNANDLQLRKGDEYFILEESNLPWWRARDKNGQEGYIPS NYVTEAEDSIEMYEWYSKHMTRSQAEQLLKQEGKEGGFIVRDSSKAGKYTVSVFAKSTGD PQGVIRHYWCSTPQSQYYLAEKHLFSTIPELINYHQHNSAGLISRLKYPVSQQNKNAPS TAGLGYGSWEIDPKDLTFLKELGTGQFGWKYGKWRGQYDVAIKMIKEGSMSEDEFIEEA KVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGCLLNYLREMRHRFQTQQLLEMCKDVC EAMEYLESKQFLHRDLAARNCLVNDQGVVKVSDFGLSRYVLDDEYTSSVGSKFPVRWSPP EVLMYSKFSSKSDIWAFGVLMWEIYSLGKMPYERFTNSETAEHIAQGLRLYRPHLASEKV YTIMYSCWHEKADERPTFKILLSNILDVMDEES

>sp|Q06187-2|BTK_HUMAN Isoform BTK-C of Tyrosine-protein kinase BTK OS=Homo sapiens OX=9606 GN=BTK

Reference: Uniprot (https://www.uniprot.org/uniprot/Q08187#sequences) SEQ ID No. 4: TEC Kinase

MNFNTILEEILIKRSQQKKKTSPLNYKERLFVLTKSMLTYYEGRAEKKYRKGFIDVSKIK CVEIVKNDDGVIPCQNKYPFQVVHDANTLYIFAPSPQSRDLWVKKLKEEIKNNNNIMIKY HPKFWTDGSYQCCRQTEKLAPGCEKYNLFESSIRKALPPAPETKKRRPPPPIPLEEEDNS EEIVVAMYDFQAAEGHDLRLERGQEYLILEKNDVHWWRARDKYGNEGYIPSNYVTGKKSN NLDQYEWYCRNMNRSKAEQLLRSEDKEGGFMVRDSSQPGLYTVSLYTKFGGEGSSGFRHY HIKETTTSPKKYYLAEKHAFGSIPEIIEYHKHNAAGLVTRLRYPVSVKGKNAPTTAGFSY EKWEINPSELTFMRELGSGLFGWRLGKWRAQYKVAIKAIREGAMCEEDFIEEAKVMMKL THPKLVQLYGVCTQQKPIYIVTEFMERGCLLNFLRQRQGHFSRDVLLSMCQDVCEGMEYL ERNSFIHRDLAARNCLVSEAGVVKVSDFGMARYVLDDQYTSSSGAKFPVKWCPPEVFNYS RFSSKSDVWSFGVLMWEVFTEGRMPFEKYTNYEVVTMVTRGHRLYQPKLASNYVYEVMLR CWQEKPEGRPSFEDLLRTIDELVECEETFGR

>sp|P42680|TEC_HUMAN Tyrcsine-prctein kinase Tec OS=Hcmc sapiens OX=9606 GN=TEC PE=1 SV=2

Reference: Uniprot (https://www.uniprot.Org/uniprot/P42680#sequences1

SEQ ID No. 5: ITK Kinase

MNNFILLEEQLIKKSQQKRRTSPSNFKVRFFVLTKASLAYFEDRHGKKRTLKGSIELSRI KCVEIVKSDISIPCHYKYPFQVVHDNYLLYVFAPDRESRQRWVLALKEETRNNNSLVPKY HPNFWMDGKWRCCSQLEKLATGCAQYDPTKNASKKPLPPTPEDNRRPLWEPEETWIALY DYQTNDPQELALRRNEEYCLLDSSEIHWWRVQDRNGHEGYVPSSYLVEKSPNNLETYEWY NKSISRDKAEKLLLDTGKEGAFMVRDSRTAGTYTVSVFTKAVVSENNPCIKHYHIKETND NPKRYYVAEKYVFDSIPLLINYHQHNGGGLVTRLRYPVCFGRQKAPVTAGLRYGKWVIDP SELTFVQEIGSGQFGLVHLGYWLNKDKVAIKTIREGAMSEEDFIEEAEVMMKLSHPKLVQ LYGVCLEQAPICLVFEFMEHGCLSDYLRTQRGLFAAETLLGMCLDVCEGMAYLEEACVIH RDLAARNCLVGENQVIKVSDFGMTRFVLDDQYTSSTGTKFPVKWASPEVFSFSRYSSKSD VWSFGVLMWEVFSEGKIPYENRSNSEWEDISTGFRLYKPRLASTHVYQIMNHCWKERPE DRPAFSRLLRQLAEIAESGL

>sp|Q08881 |ITK_HUMAN Tyrcsine-prctein kinase ITK/TSK OS=Hcmc sapiens OX=9606 GN=ITK PE=1 SV=1

Reference : Uniprct (https://www.uniprot.Org/uniprot/Q08881#sequences1

SEQ ID No. 6: TXK Kinase

MILSSYNTIQSVFCCCCCCSVQKRQMRTQISLSTDEELPEKYTQRRRPWLSQLSNKKQSN TGRVQPSKRKPLPPLPPSEVAEEKIQVKALYDFLPREPCNLALRRAEEYLILEKYNPHWW

KARDRLGNEGLIPSNYVTENKITNLEIYEWYHRNITRNQAEHLLRQESKEGAFIVRDSRH LGSYTISVFMGARRSTEAAIKHYQIKKNDSGQWYVAERHAFQSIPELIWYHQHNAAGLMT RLRYPVGLMGSCLPATAGFSYEKWEIDPSELAFIKEIGSGQFGVVHLGEWRSHIQVAIKA INEGSMSEEDFIEEAKVMMKLSHSKLVQLYGVCIQRKPLYIVTEFMENGCLLNYLRENKG KLRKEMLLSVCQDICEGMEYLERNGYIHRDLAARNCLVSSTCIVKISDFGMTRYVLDDEY VSSFGAKFPIKWSPPEVFLFNKYSSKSDVWSFGVLMWEVFTEGKMPFENKSNLQWEAIS EGFRLYRPHLAPMSIYEVMYSCWHEKPEGRPTFAELLRAVTEIAETW

>sp|P42681 |TXK_HUMAN Tyrosine-protein kinase TXK OS=Homo sapiens OX=9606 GN=TXK PE=1 SV=3

Reference: Uniprot (https://www.uniprot.Org/uniprot/P42681#sequences)

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All documents mentioned in this specification are incorporated herein by reference in their entirety.

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Claims

Claims:

1. A compound of formula (I):

and salts, solvates and protected forms thereof, wherein:

-L- is a covalent bond or Ci-e alkylene;

-L^7B- is a covalent bond or selected from Ci-e alkylene, C2-6 alkenylene,

C2-6 alkynylene and C2-6 heteroalkylene; and

2. The compound of claim 1 , wherein -L^7A- is a covalent bond, -NH- or -N(R^N)-.

3. The compound of claim 1 or claim 2, wherein -L^7B- is a covalent bond or C2-6 alkenylene.

4. The compound of any one of the preceding claims, wherein -R^7A is selected from optionally substituted aryl and optionally substituted heterocyclyl.

5. The compound of claim 4, wherein each optional substituent is a group -R^s, and where -R^s is a substituent to a carbon atom within the group -R^7A, the group -R^s is -R^sc; and

where -R^s is a substituent to a carbon atom within the group -R^7A, the group -R^s is

-R^SN; where -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 where -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; and

6. The compound of any of claims 1 to 5, having the structure:

7. A compound of formula (II):

and salts, solvates and protected forms thereof, wherein:

-L- is a covalent bond or Ci-e alkylene;

-D is an acceptor group, such as a Michael acceptor group; and

-R⁶ is -|_^6A-|_^6B-R^6A, where

-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)₂NH-, *-S(0)₂N(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,

C_2-6 alkynylene and C_2-6 heteroalkylene; and

-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, and the compound of formula (II) is not either of the following compounds:

8. The compound of claim 7, wherein -L^6A- is a covalent bond, -NH- or -N(R^N)-.

9. The compound of claim 7 or claim 8, wherein -L^6B- is a covalent bond or

C_2-6 alkenylene.

10. The compound of any one of claims 7 to 9, wherein -R^6A is optionally substituted heterocyclyl.

11. The compound of claim 10, wherein each optional substituent is -R^s, and

IMM - BMX Inhibitors 007657513 where -R^s is a substituent to a carbon atom within the group -R^6A, the group -R^s is -R^sc; and

where -R^s is a substituent to a carbon atom within the group -R^6A, the group -R^s is

-R^SN; where -R^sc is independently selected from -L^sc-R^ss, halo, hydroxy (-OH), amino (-NH2), thiol (-SH), cyano, nitro, and carboxy (-COOH), where:

12. The compound of any one of claims 7 to 11 , having the structure:

13. The compound of any one of claims 1-6 and 8-1 1 , wherein -A- is optionally substituted arylene.

14. The compound of claim 13, wherein -A- is selected from phenylene, pyridinylene, indolylene, isoindolylene, benzoimidazolylene, indolinylene, isoindolinylene,

tetrahydroquinolinylene and tetrahydroisoquinolinylene.

15. The compound of claim 13, wherein -A- is selected from phenylene and pyridinylene.

IMM - BMX Inhibitors 007657513

16. The compound of any one of claims 1-6 and 8-15, wherein -A- is not further substituted, is further monosubstituted with -R^A or is further disubstituted with -R^A, such as further monosubstituted with -R^A.

17. The compound of any one of the preceding claims, wherein -R^A is independently selected from -L^M-R^M, halo, hydroxy (-OH), amino (-NH2), 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.

18. The compound of claim 17, wherein -R^A is alkyl.

19. The compound of claim 18, wherein -R^A is methyl.

20. The compound of any one of the preceding claims, wherein -L- is a covalent bond.

21. The compound of any one of the preceding claims, wherein -D is -X-M, where

-X- is a covalent bond or -L^M-,

where -L^M- 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 -L-; and

-M is selected from alkenyl, alkynyl, heterocyclyl, alkyl substituted with cyano, and cyano.

22. The compound of claim 21 , wherein -D is -N(H)C(0)CHCH₂.

23. A pharmaceutical composition comprising a compound of formula (I) or a compound of formula (II) according to any one of claims 1 to 22, together with a pharmaceutically acceptable excipient.

24. A compound of formula (I) or a compound of formula (II) according to any one of claims 1 to 22, or a pharmaceutical composition comprising a compound of formula (I) or a compound of formula (II) according to claim 23, for use in a method of treatment.

IMM - BMX Inhibitors 007657513

25. A compound of formula (I) or a compound of formula (II) according to any one of claims 1 to 22, or a pharmaceutical composition comprising a compound of formula (I) or a compound of formula (II) according to claim 23, for use in a method of treating cancer.

26. A compound of formula (I) or a compound of formula (II) according to any one of claims 1 to 22, or a pharmaceutical composition comprising a compound of formula (I) or a compound of formula (II) according to claim 23, for use in a method of treating an

autoimmune disease.

IMM - BMX Inhibitors 007657513