EP4619376A2 - Échafaudages de concepteur cyclique pour le ciblage covalent de protéines par addition de michael - Google Patents

Échafaudages de concepteur cyclique pour le ciblage covalent de protéines par addition de michael

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Publication number
EP4619376A2
EP4619376A2 EP23841041.9A EP23841041A EP4619376A2 EP 4619376 A2 EP4619376 A2 EP 4619376A2 EP 23841041 A EP23841041 A EP 23841041A EP 4619376 A2 EP4619376 A2 EP 4619376A2
Authority
EP
European Patent Office
Prior art keywords
alkyl
aryl
alkenyl
alkynyl
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23841041.9A
Other languages
German (de)
English (en)
Inventor
Attila REMÉNYI
Tibor SOÓS
Ádám Levente PÓTI
Dániel BÁLINT
Anita ALEXA
Péter Dániel SOK
Lili TORDA
Szilárd VARGA
Kristóf OZSVÁTH
Krisztián ALBERT
Roberta PALKÓ
Orsolya EMBER
Eszter KÁLLAINÉ SZARKA
Tímea IMRE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Protenic Fejleszto Es Szolgaltato Kft
Original Assignee
Protenic Fejleszto Es Szolgaltato Kft
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Filing date
Publication date
Priority claimed from HUP2200449A external-priority patent/HU231689B1/hu
Priority claimed from HU2300245A external-priority patent/HUP2300245A1/hu
Application filed by Protenic Fejleszto Es Szolgaltato Kft filed Critical Protenic Fejleszto Es Szolgaltato Kft
Publication of EP4619376A2 publication Critical patent/EP4619376A2/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/74—Esters of carboxylic acids having an esterified carboxyl group bound to a carbon atom of a ring other than a six-membered aromatic ring
    • C07C69/757—Esters of carboxylic acids having an esterified carboxyl group bound to a carbon atom of a ring other than a six-membered aromatic ring having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/28—Radicals substituted by singly-bound oxygen or sulphur atoms
    • C07D213/30—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/55—Acids; Esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • C07D215/40—Nitrogen atoms attached in position 8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
    • C07D249/06—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles with aryl radicals directly attached to ring atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/30—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D263/32—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/06—Systems containing only non-condensed rings with a five-membered ring
    • C07C2601/10—Systems containing only non-condensed rings with a five-membered ring the ring being unsaturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/36—Systems containing two condensed rings the rings having more than two atoms in common
    • C07C2602/44—Systems containing two condensed rings the rings having more than two atoms in common the bicyclo ring system containing eight carbon atoms

Definitions

  • curcumin from turmeric, Curcuma longa
  • 4-hydroxyderricin from Angelica keiskei
  • curcumin from turmeric, Curcuma longa
  • 4-hydroxyderricin from Angelica keiskei
  • an acyclic (or open-chain) a,p-unsaturated ketone that alkylates cysteines.
  • antitumor or anti-inflammatory herbal compounds such as Withaferin A (from winter cherry, Withania somnifera) or zerumbone (from ginger, Zingiber zerumbet) contain cyclic a,P-unsaturated ketones and react with nucleophilic residues (e.g., cysteines) of proteins (vimentin, NFKB or Keapl, HuR, respectively).
  • nucleophilic residues e.g., cysteines
  • cysteine targeting covalent drug is composed of a covalent warhead and a directing moiety: the former is used as a simple nonselective chemical warhead “appendage” (e.g., acrylamide), while the latter is a structurally more complex moiety that forms specific noncovalent contacts with the target protein.
  • a covalent warhead is a modular, often transferable fragment of a more complex compound, where the former contributes to protein binding affinity by forming a covalent bond with the protein (reversible or irreversible). Its contribution to binding energy is distinct from those provided by noncovalent contacts between the protein and the small molecule.
  • This design approach were successful in many cases which is reflected by the evolution of several approved and experimental drugs that bear a,p-unsaturated carbonyls where the electrophilic beta carbon is reacted with the thiol group of specific cysteines (Jackson et al., 2017).
  • the enhanced and uncontrolled reactivity of the Michael acceptors may lead to adverse (off-target) effects by reacting with undesired proteins containing a cysteine. These unwanted effects may lead to increased idiosyncratic drug reactions and therefore these inhibitors might increase the risk of side-effects.
  • An additional problem is that cells contain free thiol nucleophiles (e.g., glutathione/GSH) in a concentration that is often many orders of magnitude higher than any targeted SH functionality (Krishnan et al., 2014).
  • this competitive reactivity may also make electrophilic warheads ineffective, since reduced glutathione is present in millimolar amounts in the cell (e.g., GSH; 1-10 mM) (Meister et al., 1988) (Resnick et al., 2019). Accordingly, significant efforts have been directed to tune the reactivity of Michael acceptors to maintain the adduct forming capacity with the target but minimize/avoid off-target and GSH reactions.
  • the acrylamide warhead has become widely used to target cysteines because it is weakly electrophilic and requires prior docking of the ligand in the proximity to the cysteine residue for covalent bond formation.
  • cellular signaling may also be inhibited by blocking the protein-protein interactions (PPI) of kinases.
  • PPI protein-protein interactions
  • MAPKs mitogen-activated protein kinases
  • a shallow PPI surface which is used by activators and substrates for binding
  • MAPK D(ocking)-groove by short linear motifs located in their unstructured regions
  • the D- groove has a conserved cysteine in all MAPKs (e.g., C161 in ERK2, C162 inp38a, and C163 in JNK1) and is comprised of a negatively charged area (referred to as the CD groove) and three small hydrophobic pockets (Garai et al., 2012).
  • Mitogen-activated protein kinases are ubiquitous regulators of cellular physiology. Increased extracellular signal-regulated kinase (ERK1/2) activity is a hallmark in cancer due to its pivotal role in promoting cell growth (Lavoie et al, 2020). Although up-regulation of p38 or c-Jun N-terminal kinase (JNK) activity is also known to be associated with specific cancers, these kinases are better known to be associated with acute or chronic inflammation due to their pivotal role in regulating cell death (Wagner et al, 2009) (Canovas et al, 2021).
  • ERK1/2 extracellular signal-regulated kinase
  • JNK c-Jun N-terminal kinase
  • JNKs play critical roles in development and homeostasis and thus complete and sustained JNK inhibition may not be desirable (Bogoyevitch et al, 2008).
  • Jnkl Jnk2
  • Jnk3 the predominant isoforms are JNK1 and JNK2 expressed ubiquitously and JNK3 expressed primarily in the nervous system.
  • JNKs are activated by phosphorylation at their activation loop by MAPK kinases (MAP2K) such as MKK4 and MKK7 and are deactivated by MAPK phosphatases including MKP1 and MKP5.
  • MAPK kinases such as MKK4 and MKK7
  • MAPK phosphatases including MKP1 and MKP5.
  • JNKs regulate their downstream substrates by phosphorylation.
  • the c-Jun and ATF2 transcription factors are phosphorylated by JNK at Ser63/73 and Thr69/71, respectively.
  • JNKs are activated when cells are exposed to stress conditions such as osmotic stress, hypoxia, UV light, and cytokines and are activated less to exposure to growth factors. Hyperactivation of JNK signaling is common in several disease states including cancer, inflammatory, and neurodegenerative diseases.
  • JNK inhibitors Based on genetic and pharmacological studies in animal models, JNK inhibitors have anti-inflammatory and neuroprotective effects. Specific blocking of JNK activity could be particularly important in treating diseases such as Alzheimer’s disease, Parkinson’s disease, asthma, diabetes, and rheumatoid arthritis (Duong et al., 2020). Moreover, selective JNK inhibition would likely have a positive role on neuron regeneration/repair after injury (Shellino et al., 2019). Thus, JNK is an attractive target for therapeutic intervention with small molecule kinase inhibitors. As a result, various inhibitors have been developed; however, limitations are noted with these inhibitors, including lack of specificity and cell toxicity.
  • an ATP binding scaffold - developed from imatinib - was linked with an acrylamide warhead. These compounds selectively target a specific cysteine - conserved in JNK1-3 (cysteine 154 in JNK3, cysteine 116 in JNK1 and JNK2), but unique among MAPKs - through covalent bond formation (Zhang et al., 2012).
  • the inhibitors - where the most selective JNK inhibitor was called JNK-IN-8 (EP 2822935) and will be referred to as such henceforth - were shown to bind to JNK irreversibly.
  • JNK-IN-8 proved to be a promising JNK-specific kinase inhibitor, nevertheless, the irreversibility of C-S cysteine adducts - formed by an acrylamide warhead - raises concerns about potential unwanted off-target effects and about the half-life in a biological environment (e.g., various S, N nucleophiles, especially 1-10 mM GSH; Meister et al., 1988).
  • Document EP 2822935 also discloses a compound without a covalent warhead (JNK-IN-6), however, this compound exhibited an almost 100-fold less potent biochemical IC50 on JNK1, 2, and 3.
  • the structure of the compound differs from the effective compounds in that the acrylamide is replaced with an approximately isosteric propyl amide group.
  • covalent warhead endows composite drugs with new properties related to the covalent nature of binding (e.g., higher binding affinity and increased residence time). These latter seem to be transferable but the functional parts, namely the noncovalent directing and the covalent warhead moieties, must be linked in such a way that fits with target-specific requirements.
  • the acrylamide warhead for example, is used repeatedly in different inhibitors binding to various members of highly different kinase families as well as against a specific RAS GTPase mutant harboring the G12C point mutation (Liu et al., 2013) (Krishnan et al., 2014) (Miller et al, 2014) (Zhao et al., 2017) (Chaikuad et al., 2018) (Ostrem et al, 2013) (Kwiatkowski et al, 2014).
  • a reversible covalent mechanism can offer a better balance between potency and selectivity compared to irreversible mechanisms (Tuley et al, 2018).
  • reversible warheads with low off-rates/long residence time could provide an advantage as parts of proteolysis targeting chimeras (PROTAC) compared to irreversible warheads, because the warhead moiety could be recycled in the cell after target protein degradation (He et al, 2022) (Smith et al, 2019) (Guo et al, 2020) (Ishida et al, 2021).
  • PROTAC proteolysis targeting chimeras
  • Cyclopentenone or cyclohexenone derivatives had formally been synthesized and used for various synthetic transformations (Berkes et al., 2016) (Varga et al., 2020) (Oswy et al., 1994) (Oswy et al., 1996) (Kato et al., 2002) (Okano et al., 1988) (Dobly et al., 1968) (Maier et al., 1993) (Meyer et al., 2000) (Souza et al, 2002) (Hsieh et al, 2008) (Liu et al, 2005) (Li et al 2022) (Document US11518729B2), however, these studies report only on accidental examples since none of them suggest a possibility of protein targeting.
  • cyclopent-2 -en-l-one (CPN) and cyclohex-2-en-l-one (CHN) form a stable covalent adduct with soft nucleophiles such as GSH, V-acetylcysteine (NAC) and thiols from some model proteins (bovine serum albumin, glyceraldehyde-3- phosphate dehydrogenase, creatine kinase, papain, Keapl) (Sauerland et al, 2021).
  • Deny J.L. et al have produced the following bis-Michael acceptor compound: which was suggested to be able to target Cysl51 in Keapl and thus increased Nrf2 -mediated transcription.
  • the monovalent version was far less potent (Deny et al, 2016; 7).
  • the methyl groups at C4 were introduced to increase steric hindrance at this position, because a compound lacking substitutions at C4 were deemed to be too electrophilic, and a dioxolane with two electron withdrawing moieties at this position was found also not useful. Compounds with electronic withdrawing groups at C4 thus were then excluded from their analysis and other electron withdrawing groups (EWG) were not exploited to fine-tune amino acid adduct formation.
  • EWG electron withdrawing groups
  • the applied warheads display a rather limited structural variance and complexity (e.g., simple open-chain, acyclic acrylamides) which automatically limits the attainable chemical space.
  • the applied reactive warheads are added appendages in the late-stage of the synthetic route, thus a warhead scaffold that can be synthetically easily varied using orthogonal chemistry and used as a tunable covalent warhead is still missing.
  • Such a structurally more complex scaffold would be much more like the warheads of the natural products and is expected to be more selective in targeting nucleophiles found in proteins.
  • a more complex scaffold might be more suitable for targeting shallow protein surfaces involved in protein-protein interactions.
  • more complex warheads for example cyclic scaffolds, for example with 5- or 6-membered rings, distinct from open-chain acrylamide-based warheads, may also be used as specific anchors linked to bigger directing moieties (noncovalent inhibitors) to further enhance specificity and/or residency time of the composite drug.
  • a structurally complex, reversible covalent warhead with the possibility of functionalization at several positions would be useful to increase the binding strength of noncovalent fragments/inhibitors that bind within -10-20 A from a targetable amino acid; for example, when the warhead is linked to a noncovalent moiety using an appropriate linker.
  • the basis of the present invention is cyclic, sterically and electronically fine-tuned Michael acceptor-based chemical warheads forming a reversible covalent bond to specific surface amino acids on proteins.
  • additional critical structural features of these structures are the double activated olefinic group in the a-position and double substitution in y-position with appropriate electron withdrawing group(s) allowing the fine-tuning of steric and electronic properties.
  • the special structure of these new warheads allows far better control over the electronic and steric properties influencing covalent bond formation to specific amino acids compared to previously utilized acyclic acrylamides.
  • these tailored warheads mainly target the cysteine, but also other nucleophilic amino acids, e.g., histidine, thus the adduct serves as a covalent anchor capable of increasing the binding affinity and decreasing the off-rate.
  • these reversible warheads provide an alternative to the practice of using an irreversible electrophile, particularly if one considers that unwanted crossreactions in the cell are likely to be more devastating if happen through irreversible covalent bond formation.
  • the cyclic and chiral nature of the scaffold is another distinct feature offering a further advantage: the local and inherently chiral environment of specific nucleophiles can be better exploited in creating specific inhibitors, for example by varying the configuration of substituent groups within the cyclic warhead scaffold.
  • the present invention provides compounds of Formula (I), and pharmaceutically acceptable salts and compositions thereof.
  • the present invention further provides methods of using the inventive warhead scaffolds targeting preferably, but not limited to cysteine or histidine, more preferably cysteine, compounds, and pharmaceutically acceptable salts and compositions thereof, to study the inhibition of proteins, by affecting protein levels as parts of proteolysis targeting chimeras (PROTAC) or by affecting protein activity as protein-protein interaction or enzymatic activity inhibitors, particularly kinases, more specifically, but not limited to mitogen-activated protein kinases (MAPKs), and such compounds for use as therapeutics in the prevention and treatment of diseases associated with proteins containing a targetable cysteine or other nucleophilic amino acid, e.g., histidine, for example, but not limited to, kinases or MAPKs.
  • a targetable cysteine or other nucleophilic amino acid e.g., histidine
  • MAPKs mitogen-activated protein kinases
  • the inventive cysteine (or other nucleophilic amino acid, e.g., histidine) modifying warhead scaffolds, compounds are used for the prevention and treatment of proliferative diseases (e.g., cancer), neurodegenerative diseases, metabolic disorders, inflammatory diseases, and cardiovascular diseases.
  • proliferative diseases e.g., cancer
  • neurodegenerative diseases e.g., metabolic disorders, inflammatory diseases, and cardiovascular diseases.
  • R 2 is selected from C1.4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-7 carbocyclyl, 3-7 membered heterocyclyl, Ce-io aryl, 5- 7 membered heteroaryl, halo-Ci.4 alkyl, aryl-Ci.4 alkyl, -C(O)OH, -C(O)O-Ci-4 alkyl, -C(O)O-C2-4 alkenyl, -C(O)O-C2-4 alkynyl, -C(0)0-C 6 -io aryl, -C(O)O-[(CH 2 ) 2 O] n -Ci-4 alkyl, -C(O)O-[(CH 2 ) 2 O] n -C2-4 alkenyl, -C(O)O-[(CH 2 ) 2 O] n -C2-4 alkenyl, -C(O)O-[(CH 2 ) 2
  • R 3 is selected from -C(O)OH, -C(O)O-Ci-4 alkyl, -C(O)O-C 2 .4 alkenyl, -C(O)O-C 2 .4 alkynyl, -C(0)0-Ce-io aryl, - C(O)O-[(CH 2 ) 2 O] n -Ci- 4 alkyl, -C(O)O-[(CH 2 ) 2 O] n -C 2.4 alkenyl, -C(O)O-[(CH 2 ) 2 O] n -C 2.4 alkynyl, -C(O)O-[(CH 2 ) 2 O] n - C(O)OH, -C(O)O-[(CH 2 ) 2 O] n -C(O)OCi- 4 alkyl, -C(O)O-[(CH 2 ) 2 O] n -C(O)O-
  • R 2 and R 3 may be combined together with the atom to which they are attached to form a C4-6 carbocyclyl or 4-6 membered heterocyclyl
  • X is -CHR 4 - or -CHR 4 -CHR 5 - or direct bond, preferably -CHR 4 - or -CHR 4 -CHR 5 -, wherein
  • R 4 is H, hydroxyl, C1.4 alkyl, CM alkenyl,
  • R 4 and R 5 may be combined together with the atoms to which they are attached to form a C3-7 carbocyclyl or 3-7 membered heterocyclyl
  • R 6 is Ce-io aryl, 5-7 membered heteroaryl, halo-Ci.4 alkyl, aryl-Ci.4 alkyl, -C(O)O-Ci-4 alkyl, -C(O)O-C 2 .4 alkenyl, -C(O)O-C 2-4 alkynyl, -C(0)0-C 6 -io aryl, -C(O)-Ci- 4 alkyl, -C(O)O-Ci- 4 alkyl-C 6 -io aryl, -C(O)-C 2.4 alkenyl, -C(O)-C 2.4 alkynyl, -C(0)-C 6 -io aryl, -C(O)NH 2 , -C(O)NHCI- 4 alkyl, -C(O)NHC 2.4 alkenyl, -C(O)NHC 2.4 alkynyl, -C
  • R 6 is composed of 2-10 moieties selected from -C(O)-, -NH-, -N(CH 3 )-, -O-, -CH 2 -, optionally substituted -Ce-io aryl, optionally substituted 3-7 membered carbocyclyl, optionally substituted 3-7 membered heterocyclyl, optionally substituted 5-7 membered heteroaryl, resulting in a chemically reasonable radical;
  • R 7 is H, Ce-io aryl, 5-7 membered heteroaryl, vinyl, -[(CH 2 ) 2 O] n -Ci-4 alkyl, -[(CH 2 ) 2 O] n -C 2 .4 alkenyl, -[(CH 2 ) 2 O] n - C 2-4 alkynyl, -[(CH 2 ) 2 O] n -C(O)OH, -[(CH 2 ) 2 O] n -C(O)OCi- 4 alkyl, -[(CH 2 ) 2 O] n -C(O)O-C 2.4 alkenyl, -[(CH 2 ) 2 O] n -C(O)O- C 2-4 alkynyl, -[(CH 2 ) 2 O] n -C(O)NH 2 , -[(CH 2 ) 2 O] n -C(O)NHCi- 4 alkyl,
  • R 8 is 5-6-membered heterocyclyl
  • the heterocyclyl is optionally substituted with one or more substituent independently selected from C1.4 alkyl, C 2 .4 alkenyl, C 2 .4 alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, Ce-io aryl, 5-10 membered heteroaryl, where the carbocyclyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one or more substituent independently selected from C1.4 alkyl, halogen, hydroxyl, -OC1.4 alkyl, -NH 2 , -NHC1.4 alkyl, - N(CI-4 alkyl) 2 , or 5-10 membered heteroaryl optionally substituted with 1 or 2 Ci- 2 alkyl; halo-Ci.4 alkyl, aryl-Ci.4 alkyl, -C(O)O-Ci-4 alkyl, -C(O)O-C 2
  • Xaan is an apolar residue, preferably Leu, He or Ala, preferably Leu
  • Xaaw is an apolar residue, preferably Leu, He or Ala, preferably Ala
  • Xaag is a residue comprising an amide or a basic residue, preferably Arg or Lys
  • Xaa? is a residue comprising an amide or a basic residue, preferably Arg or Lys
  • Xaa? is a residue comprising an amide or a basic residue, preferably Arg or Lys, wherein at least two of Xaag Xaa? Xaa? is a basic residue, preferably Arg or Lys, Xaa? is an apolar residue, preferably Leu, He or Ala, preferably Ala, Xaa? is an apolar residue, preferably Leu, He or Ala, preferably Leu,
  • Xaai is a residue comprising an amide or a basic residue, preferably Arg or Lys,
  • Xaa? is a polar residue, preferably Ser or Thr,
  • R 1 is H, Ci-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, Ce-io aryl or 5-7 membered heteroaryl, preferably H or C1.3 alkyl, C2-3 alkenyl, C2-3 alkynyl, Ce-io aryl, more preferably H, C1.3 alkyl, or phenyl;
  • R 2 is selected from C1.4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-7 carbocyclyl, 3-7 membered heterocyclyl, Ce-io aryl, 5- 7 membered heteroaryl, halo-Ci.4 alkyl, aryl-Ci.4 alkyl, -C(O)OH, -C(O)O-Ci-4 alkyl, -C(O)O-C2-4 alkenyl, -C(O)O-C2-4 alkynyl, -C(0)0-C 6 -io aryl, -C(O)O-[(CH 2 ) 2 O] n -Ci-4 alkyl, -C(O)O-[(CH 2 ) 2 O] n -C2-4 alkenyl, -C(O)O-[(CH 2 ) 2 O] n -C2-4 alkenyl, -C(O)O-[(CH 2 ) 2
  • R 3 is selected from -C(O)OH, -C(0)0-CM alkyl, -C(O)O-C 2 .4 alkenyl, -C(O)O-C 2 .4 alkynyl, -C(0)0-Ce-io aryl, - C(O)O-[(CH 2 ) 2 O] n -Ci- 4 alkyl, -C(O)O-[(CH 2 ) 2 O] n -C 2 .4 alkenyl, -C(O)O-[(CH 2 ) 2 O] n -C 2 .4 alkynyl, -C(O)O-[(CH 2 ) 2 O] n - C(O)OH, -C(O)O-[(CH 2 ) 2 O] n -C(O)OCi- 4 alkyl, -C(O)O-[(CH 2 ) 2 O] n -C(O)O-C
  • R 2 and R 3 may be combined together with the atom to which they are attached to form a C4-6 carbocyclyl or 4-6 membered heterocyclyl
  • X is -CHR 4 - or -CHR 4 -CHR 5 - or direct bond, preferably -CHR 4 - or -CHR 4 -CHR 5 -, wherein
  • R 4 is H, hydroxyl, CM alkyl, CM alkenyl,
  • R 4 and R 5 may be combined together with the atoms to which they are attached to form a C3-7 carbocyclyl or 3-7 membered heterocyclyl
  • R 6 is C3-7 carbocyclyl, 3-7 membered heterocyclyl, Ce-io aryl, 5-7 membered heteroaryl, halo-Ci.4 alkyl, aryl-Ci.4 alkyl, -C(0)0-CM alkyl, -C(O)O-C 2.4 alkenyl, -C(O)O-C 2.4 alkynyl, -C(0)0-C 6 -io aryl, -C(0)-CM alkyl, -C(0)0-CM alkyl-Ce-ioaryl, -C(O)-C 2.4 alkenyl, -C(O)-C 2.4 alkynyl, -C(0)-C 6 -io aryl, -C(O)NH 2 , -C(O)NHCM alkyl, -C(0)NHCM alkenyl, -C(0)NHCM alkynyl, -C(O)NHCM alkyl-OH,
  • R 6 is composed of 2-10 moieties selected from -C(O)-, -NH-, -N(CH 3 )-, -O-, -CH 2 - optionally substituted -Ce-io aryl, optionally substituted 3-7 membered carbocyclyl, optionally substituted 3-7 membered heterocyclyl, optionally substituted 5-7 membered heteroaryl, resulting in a chemically reasonable radical;
  • R 7 is H, Ce-ioaryl, 5-7 membered heteroaryl, vinyl, -[(CH 2 ) 2 O] n -Ci.4 alkyl, -[(CH 2 ) 2 O] n -C 2 .4 alkenyl, -[(CH 2 ) 2 O] n - CM alkynyl, -[(CH 2 ) 2 O] n -C(O)OH, -[(CH 2 ) 2 O] n -C(O)OCi- 4 alkyl, -[(CH 2 ) 2 O] n -C(O)O-C 2 -4 alkenyl, -[(CH 2 ) 2 O] n -C(O)O- CM alkynyl, -[(CH 2 ) 2 O] n -C(O)NH 2 , -[(CH 2 ) 2 O] n -C(O)NHCi- 4 alkyl, -
  • R 8 is 5-6-membered heterocyclyl
  • the heterocyclyl is optionally substituted with one or more substituent independently selected from CM alkyl, CM alkenyl, CM alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, Ce-io aryl, 5-10 membered heteroaryl
  • the carbocyclyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one or more substituent independently selected from CM alkyl, halogen, hydroxyl, -OCM alkyl, -NH 2 , -NHCM alkyl, -N(CM alkyl) 2 or 5-10 membered heteroaryl; halo-Ci.4 alkyl, aryl-Ci.4 alkyl, -C(O)O-CM alkyl, -C(0)0-CM alkenyl, -C(O)O- Ce-io aryl, -C(0)-CM alkyl
  • LARRRALRSKS* where * means the N terminal of the peptide.
  • R 1 is H, Ci-4 alkyl, Ce-io aryl, preferably H, C1.3 alkyl, phenyl, most preferably H
  • R 2 is selected from C1.4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-7 carbocyclyl, 3-7 membered heterocyclyl, Ce-io aryl,
  • R 3 is selected from -C(O)OH, -C(O)O-Ci-4 alkyl, -C(O)O-C2-4 alkenyl, -C(O)O-C2-4 alkynyl, -C(0)0-Ce-io aryl, - C(O)O-[(CH 2 ) 2 O] n -C(O)OH, -C(O)O-[(CH 2 )2O] n -C(O)OCi- 4 alkyl, -C(O)O-[(CH 2 )2O] n -C(O)NH 2 , -C(O)O-[(CH 2 ) 2 O] n - C(O) NHC1-4 alkyl, -C(O)O-[(CH 2 ) 2 O] n -C(O) NC1.4 alkyl 2 , -C(O)O-[(CH 2 )2O] n -NH 2 , -
  • R 6 is 3-7 membered heterocyclyl, Ce-io aryl, 5-7 membered heteroaryl, -C(O)O-Ci-4 alkyl, -C(O)O-C2-4 alkenyl, - C(O)O-C 2 -4 alkynyl, -C(0)0-C 6 -io aryl, -C(O)O-Ci- 4 alkyl-C 6 -io aryl, -C(O)NH 2 , -C(O)NHCI- 4 alkyl, -C(O)NHC 2 -4 alkenyl, -C(O)NHC 2 -4 alkynyl, -C(O)NCI- 4 alkyl 2 , -C(O)NC 2 -4 alkenyl 2 , -C(O)NC 2 -4 alkynyl 2 , -C(O)NHCI- 4 alkyl-OH, - C(O)NH-CI-4 al
  • R 9 is -OH, -NH 2 , -OCI-4 Alkyl, -Ci- 4 Alkyl, Halogen, -CN
  • Y is 0-3, preferably 0-1
  • Z is CH, N or NH as valency permits
  • X is -CHR 4 - or -CHR 4 -CHR 5 - or direct bond, preferably -CHR 4 -CHR 5 - wherein
  • R 5 is H, C1.3 alkyl, C2-4 alkenyl, or R 5 together with R 3 may form a -CH2-CXCH2)- group
  • R 7 is H, Ce-io aryl, 5-7 membered heteroaryl, the aryl and heteroaryl are optionally substituted with one or more substituent independently selected from C1.4 alkyl, halogen, -NH 2 , -NHC1.4 alkyl, -N(CI-4 alkyl) 2 ; vinyl, -[(CH 2 )2O] n - C(O)OH, -[(CH 2 )2O] n -C(O)OCi- 4 alkyl, -[(CH 2 )2O] n -C(O)NH 2 , -[(CH 2 )2O] n -C(O)NHCi.
  • R 8 is 5-6-membered heterocyclyl, the heterocyclyl is optionally substituted with one or more substituent independently selected from CM alkyl, C 2 .4 alkenyl, C 2 .4 alkynyl, C3-6 carbocyclyl, 3-6 membered heterocyclyl, Ce-8 aryl, 5-10 membered heteroaryl, where the carbocyclyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one or more substituent independently selected from CM alkyl, halogen, hydroxyl, -OC1.4 alkyl, -NH 2 , -NHC1.4 alkyl, -N(Ci- 4 alkyl) 2 or 5-10 membered heteroaryl; halo-Ci.4 alkyl, aryl-Ci.4 alkyl, -C(O)O-Ci-4 alkyl, -C(O)O-C 2 .4 alkenyl, -C(0)0-Ce-io
  • W is one of the following
  • R 1 is H or methyl, Ce aryl, preferably H.
  • R 2 is selected from C1.3 alkyl, C2-3 alkenyl, C2-3 alkynyl; carbocycle selected from C3-6 cycloalkyl groups, preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; heterocycle selected from 3-7 membered heterocyclyl, preferably 4-6 membered heterocyclyl, more preferably azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, pyranyl, morpholinyl, oxazinyl, dioxanyl; Ce-ioaryl, 5-7 membered heteroaryl, halo-Ci-salkyl, Ce
  • R 3 is selected from -C(O)O-Ci- 4 alkyl, -C(O)O-C 2 -3alkenyl, -C(O)O-Ci- 4 alky 1-OCR 7 , -C(O)O-Ci- 3 alkyl-R 8 , -C(O)O- Ce-io aryl, -C(O)-Ci-3 alkyl, -C(O)-C2-3 alkenyl, -C(O)-C2-3 alkynyl, -C(O)-Ce aryl, and
  • R 7 is H, Ce-8 aryl, 5-6 membered heteroaryl, the aryl, heteroaryl optionally substituted with one or more substituents independently selected from C1.2 alkyl, halogen, hydroxyl, -OCi- 4 alkyl, -NH 2 , -NHC1.2 alkyl, -N(CI-2 alkyl) 2
  • R 8 is 5-6-membered heteroaryl, preferably the heteroaryl comprises 1-3 N atoms, the heteroaryl is optionally substituted with Ce aryl, the aryl is optionally substituted with one or more substituent independently selected from C1.2 alkyl, halogen, hydroxyl, -OCi- 4 alkyl, -NH 2 , -NHC1.2 alkyl, -N(CI-2 alkyl) 2 or 5-10 membered heteroaryl; C1.3 alkyl, C2-3 alkenyl, C2-3 alkynyl, halo-Ci-salkyl, -C(O)NH 2 , -C(O)NHCI-3 alkyl, -C(O)NCI-3 alkyl 2 , -C1.3 alkyl-C(O)O-Ci-3 alkyl, - C1-3 alkyl-C(O)OH, -C1.3 alkyl-C(O)O-C 2-3 alkenyl, -C1.3 al
  • R 7 is H, Ce-io aryl, 5-6 membered heteroaryl, the aryl, heteroaryl optionally substituted with one or more substituents independently selected from -NH 2 , -NHC1.2 alkyl, -N(CI-2 alkyl) 2 , where * is the point of attachment and
  • L 1-6 or R 2 together with R 5 may form a -CH2-C(CH 2 )- group.
  • R 2 is C1.3 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 carbocyclyl, 3-6 membered heterocyclyl, Ce aryl, 5-7 membered heteroaryl, halo-Ci.3 alkyl, aryl-Ci.3 alkyl, or together with R 5 may form a -CH2-C(CH 2 )- group
  • R 3 is -C(O)O-Ci- 4 alkyl, -C(O)O-C 2.4 alkenyl, -C(O)O-C 2.4 alkynyl, -C(0)0-C 6 -io aryl, -C(O)-Ci- 3 alkyl, -C(O)-C 2 .
  • X is -CHR 4 - or -CHR 4 -CHR 5 -, wherein
  • R 4 is H, methyl
  • R 5 is H, or together with R 2 may form a -CH2-C(CH 2 )- group
  • R 5 may form a -CH2-C(CH 2 )- group
  • R 2 is independently selected from -C(O)-O-Ci-3alkyl, -C(O)-O-C2-3alkenyl, -C(O)-O-C2-3 alkynyl, -C(O)-O-aryl, -C(O)- Ci.
  • phenyl -Ci-salkyl preferably -C(O)-O- methyl, -C(O)-O-ethyl, -C(O)-O-phenyl, -C(O)-phenyl or -C(0)-0-CH 2 -OCH, -C(0)-0-CH 2 -OC-phenyl, C(O)O- CH 2 -R 8
  • R 3 is independently selected from -C(O)-O-Ci-3alkyl, -C(O)-O-C2-3alkenyl, -C(O)-O-C2-3 alkynyl, -C(O)-O-aryl, -C(O)- Ci.
  • the compound according to any of the previous points or pharmaceutically acceptable salts thereof wherein the compound of formula (I) is a compound of formula (I- 1), and wherein one of R 2 and R 3 are as defined herein, preferably
  • R 6 is as defined in any of points 1 to 9, preferably in point 3 or point 4. 9.2.
  • Re is as defined in any of points 1 to 8, preferably in point 2 or point 3.
  • R 7 is H, Ce-ioaryl, 5-7 membered heteroaryl, vinyl, -[(CH 2 ) 2 O] n -C(O)OH, -[(CH 2 ) 2 O] n -C(O)OCi-4 alkyl, - [(CH 2 ) 2 O] n -C(O)NH 2 , -[(CH 2 ) 2 O] n -C(O)NHCi- 4 alkyl, -[(CH 2 ) 2 O] n -C(O)NCi- 4 alkyl 2 -[(CH 2 ) 2 O] n -NH 2 , -[(CH 2 ) 2 O] n - NHC1-4 alkyl, -[(CH 2 ) 2 O] n -NCi- 4 alkyl 2 , -C(O)O-[(CH 2 ) 2 O] n - Ce-ioaryl, -C(O)O-[(CH 2
  • R 8 is 5-6-membered heterocyclyl
  • the heterocyclyl is optionally substituted with one or more substituent independently selected from Ci-4 alkyl, CM alkenyl, C 2 .4 alkynyl, C3-6 carbocyclyl, 3-6 membered heterocyclyl, Ce-8 aryl, 5-10 membered heteroaryl
  • the aryl and heteroaryl are optionally substituted with one or more substituent independently selected from C1.4 alkyl, halogen, -NH 2 , -NHC1.4 alkyl, -N(CI-4 alkyl) 2 ; halo-Ci-4 alkyl, aryl-Ci.4 alkyl, -C(O)O-Ci-4 alkyl, -C(O)O-C 2 .4 alkenyl, -C(0)0-Ce-io aryl, -C(O)-Ci-4 alkyl, -C(O)-C 2 .4 alkenyl,
  • R 6 is -C(O)O-methyl, -C(O)O-ethyl, -C(O)O-tertbutyl, -C(O)O-CH 2 -phenyl, -C(O)NH-teributyl, -C(O)NH- phenyl, -C(O)NH-CH 2 -phenyl, -C(O)NH-C(Me) 2 -CH 2 -OH, -C(O)NH-CH 2 -C(Me) 2 -OH, preferably -C(O)O-teributyl, - C(O)Obenzyl, 5-methyloxazol-2-yl, quinolin-8-yl.
  • Cyc A is an optionally substituted aryl or heteroaryl ring, preferably an optionally substituted phenyl or pyridine ring, more preferably an optionally substituted 3 -pyridinyl ring; even more preferably an unsubstituted 3 -pyridinyl ring;
  • Y is N or CR 10 , preferably N
  • W is CR 11 or N, preferably CR 11
  • R 10 is H or Ci-e alkyl, preferably H
  • R 11 is H or Ci-6 alkyl, preferably H is H, halogen, Ci-e alkyl, preferably H or Cl, more preferably H
  • Cyc c is phenyl optionally substituted with R 13 group(s) or a phenyl isostere, preferably
  • R 13 is halogen, Ci-e alkyl, halo-Ci-e alkyl, preferably Cl, methyl or trifluoromethyl, more preferably methyl m is an integer of 0 to 4, preferably 0 or 1
  • R 14 is H, Ci-e alkyl or N-protecting group, preferably H, methyl, ethyl, or an N-protecting group, more preferably
  • R 15 is halogen, Ci-e alkyl, halo-Ci-e alkyl, preferably C1.3 alkyl, preferably halogen, methyl, ethyl, or trifluoromethyl, preferably H or methyl, n is an integer of 0 to 4, preferably 0, 1 or 2, more preferably 0 or 1
  • Q is direct bond or -[-CHR 16 -] 0 -
  • R 16 is H or Ci-e alkyl, preferably H o is an integer of 1 or 2, preferably 1
  • Z is NH or O
  • R 6 is selected from attachment and
  • R 2 is Ci-salkyl, Cwalkenyl, Cwalkynyl, cyclohexyl, lialo-C i-,alky 1.
  • phenyl -Ci-salkyl preferably methyl, cyclohexyl, phenyl, benzyl, lialo-Ci _,alky 1.
  • -CH 2 -CH 2 -CH 2 C1, -CH 2 -CH CH 2
  • R 2 together with R 5 may form -CH 2 -C(CH 2 )-,
  • R 6 is -C(O)O'Bu, -C(O)Obenzyl.
  • binding is covalent and reversible.
  • Said binding is detectable by any assay suitable to detect protein binding of organic molecules, for example surface plasmon resonance (SPR), isothermal calorimetry (ITC), fluorescence polarization (FP) or NanoBiT assay.
  • SPR surface plasmon resonance
  • ITC isothermal calorimetry
  • FP fluorescence polarization
  • NanoBiT NanoBiT assay.
  • the Michael-acceptor compound of any of points 1 to 15 is capable of binding in particular to a Cys and/or a His of a protein, as a nucleophile.
  • the protein binding Michael-acceptor compound binds to a Cys and/or a His residue as a nucleophile. 16.1
  • the protein binding Michael-acceptor compound is a Michael-acceptor type protein inhibitor, wherein activity of a protein is inhibited by binding of the compound of the invention to said protein. In particular binding is targeted to a site of said protein which is essential to protein activity, whereas the warhead moiety of the compound of the invention covalently and reversibly binds to a nucleophile amino acid residue.
  • the nucleophile amino acid is a Cys and/or a His residue.
  • the site essential to protein activity is preferably a binding surface.
  • the binding surface is a part of the protein surface to which another protein, e.g. a binding partner, preferably a protein substrate/activator/deactivator/scaffold protein or protein domain is capable of binding which is essential to the activity of said protein to which the Michael-acceptor compound of the invention binds.
  • a binding partner e.g. a protein substrate/activator/deactivator/scaffold protein or protein domain is capable of binding which is essential to the activity of said protein to which the Michael-acceptor compound of the invention binds.
  • the site essential to protein activity is preferably an active site region, wherein a part of the compound of the invention interferes with the active site of the protein.
  • the active site region comprises an appropriate nucleophile residue, preferably a Cys and/or a His residueto which the warhead moiety of the Michael-acceptor compound of the invention binds; as well as the active site.
  • an appropriate nucleophile residue preferably a Cys and/or a His residueto which the warhead moiety of the Michael-acceptor compound of the invention binds; as well as the active site.
  • the nucleophile residue and the active site of said protein is in a sufficient proximity whereby the Michael-acceptor compound, once bound, interferes with the activity of said protein.
  • Said inhibition is detectable by any assay suitable to detect protein activity or inhibition of activity of said protein, for example biochemical enzymatic assays in vitro or cell-based assays capable of detecting protein levels or activity in cells (e.g. by western blotting using specific antibodies).
  • Cys is a conservative Cys of the protein molecule.
  • the His is a conservative His of the protein molecule.
  • Cys is not a conservative Cys of the protein molecule.
  • the His is not a conservative His of the protein molecule.
  • the protein binding Michael-acceptor compound is a (part of a) bifimctional molecule, comprising a Michael-acceptor type protein binding moiety and a further protein binding moiety capable of binding to a further protein and preferably a linker between the Michael-acceptor type protein binding molecule or moiety and the further protein binding moiety.
  • the protein binding Michael-acceptor compound is a moiety of a bifunctional molecule, comprising the Michael-acceptor type protein binding moiety.
  • the bifunctional molecule is a homobifunctional molecule wherein the protein binding function of said bifunctional molecule is identical, i.e. bind the same protein, i.e. the protein and said further protein are identical.
  • the bifunctional molecule is a heterobifunctional molecule wherein the protein binding functions of said bifunctional molecule are different, preferably bind different proteins, i.e. said protein and said further protein are different.
  • the further protein binding moiety capable of binding to a further protein is a Michael-acceptor type protein binding moiety as defined herein.
  • the further protein is a protein the function of which is to be utilized in connection with said protein.
  • the further protein is a ligase protein.
  • the further protein is a ubiquitin ligase capable of ubiquitination of said protein as a target protein.
  • the compound of the invention is a part of a proteolysis targeting chimera (PROTAC).
  • the invention provides a proteolysis targeting chimera comprising the compound of any one of points 1 to 16.1.
  • Said binding is covalent and reversible.
  • Said binding is detectable by any assay suitable to detect protein binding as defined in point 16.
  • the Michael-acceptor compound of any of points 1 to 16 is capable of binding in particular to a Cys and/or a His of a protein, as a nucleophile.
  • the protein binding Michael-acceptor compound binds to a Cys and/or a His residue as a nucleophile.
  • the invention relates to the use, preferably ex vivo use of a Michael-acceptor compound of any one of points 1 to 16 as a protein activity modulator, wherein the compound binds to a Cys and/or His residue of said protein with a reversible covalent bond.
  • binding of the Michael-acceptor compound of the invention modifies protein activity, whereas the warhead moiety of the compound of the invention covalently and reversibly binds to a nucleophile amino acid residue.
  • the nucleophile amino acid is a Cys and/or a His residue.
  • the site essential to protein activity is a binding surface.
  • the binding surface is a part of the protein surface to which another protein, e.g. a binding partner can bind.
  • the binding partner may be a protein inhibitor whereby the Michael -acceptor compound, by blocking the binding of the inhibitor, serves as an activator of said protein.
  • the binding partner is a protein agonist of said protein and the Michael-acceptor compound, by blocking the binding of the agonist, serves as an inhibitor of said protein.
  • binding surface is as defined in the previous point(s) or as defined herein.
  • the Cys is a conservative Cys of the protein to which the binding partner can bind.
  • the His is a conservative His of the protein to which the binding partner can bind.
  • Cys is not a conservative Cys of the protein to which the binding partner can bind.
  • His is not a conservative His of the protein to which the binding partner can bind.
  • the invention relates to the use (preferably ex vivo use) of a compound of any one of points 1 to 16 as a Michael-acceptor type protein inhibitor.
  • binding of the Michael-acceptor type protein inhibitor of the invention is targeted to a site of said protein which is essential to protein activity, whereas the warhead moiety of the compound of the invention covalently and reversibly binds to a nucleophile amino acid residue.
  • the nucleophile amino acid is a Cys and/or a His residue.
  • the site essential to protein activity is preferably a binding surface.
  • binding surface is as defined in the previous point(s) or as defined herein.
  • the site essential to protein activity is preferably an active site region.
  • the active site region is as defined in the previous point(s) or as defined herein.
  • Said inhibition is detectable by any assay suitable to detect protein activity or inhibition of activity of said protein, for example biochemical enzymatic assays in vitro (e.g. PhALC assay) or cell-based assays capable of detecting protein levels or activity in cells (e.g. by western blotting using specific antibodies).
  • biochemical enzymatic assays in vitro e.g. PhALC assay
  • cell-based assays capable of detecting protein levels or activity in cells (e.g. by western blotting using specific antibodies).
  • the compound binds to a conservative Cys of an ATPase domain in said protein.
  • the compound binds to a conservative His of an ATPase domain in said protein.
  • the compound binds to a conservative Cys of an ATPase active site region in said protein, preferably in proximity of an ATP binding site.
  • the compound binds to a conservative His of an ATPase active site region in said protein, preferably in proximity of an ATP binding site.
  • the Cys is not a conservative Cys.
  • the His is not a conservative His.
  • said protein is a kinase and the compound of the invention is used as a kinase inhibitor.
  • the compound is as defined in point 16.
  • the protein binding Michael-acceptor compound is used as a moiety of a bifunctional molecule, as defined in point 16.1.
  • the bifunctional molecule comprises the Michael-acceptor type protein binding moiety and a further protein binding moiety capable of binding to a further protein and preferably a linker between the Michael-acceptor type protein binding moiety and the further protein binding moiety.
  • the further protein is a ligase protein.
  • the further protein is a ubiquitin ligase capable of ubiquitination of said protein as a target protein.
  • the compound of the invention is used as a moiety, which is a part of a proteolysis targeting chimera (PROTAC).
  • PROTAC proteolysis targeting chimera
  • the one or more of the disclaimers defined in point 1.1 do not apply in the use of said compound as a kinase inhibitor.
  • disclaimer defined in point 1.1.1 the disclaimer defined in point 1.1.2, the disclaimer defined in point 1.1.3, the disclaimer defined in point 1.1.4, the disclaimer defined in point 1.1.5, the disclaimer defined in point 1.1.6, the disclaimer defined in point 1.1.7.
  • the kinase inhibitor is an inhibitor of a kinase of the family of mitogen -activated protein kinases (MAP kinases or MAPK).
  • MAP kinases mitogen -activated protein kinases
  • the kinase inhibitor is an inhibitor of a kinase of the MAPK subfamily of extracellular regulated kinases 1 (ERK1).
  • the kinase inhibitor is an inhibitor of a kinase of the MAPK subfamily of extracellular regulated kinases 2 (ERK2).
  • ERK2 extracellular regulated kinases 2
  • the kinase inhibitor is an inhibitor of a kinase of the MAPK subfamily of c-Jun N-terminal kinases (JNK).
  • JNK c-Jun N-terminal kinases
  • the kinase inhibitor is an inhibitor of a kinase of the MAPK subfamily of p38 kinases.
  • the kinase inhibitor is an inhibitor of a kinase of the MAPK subfamily of extracellular regulated kinases 5
  • the kinase is selected from the following kinases: ERK2, JNK1, JNK2, JNK3, p38alpha.
  • the one or more of the disclaimers defined in point 1.1 do not apply in the use of said compound as a kinase inhibitor.
  • disclaimer defined in point 1.1.1 the disclaimer defined in point 1.1.2, the disclaimer defined in point 1.1.3, the disclaimer defined in point 1.1.4, the disclaimer defined in point 1.1.5, the disclaimer defined in point 1.1.6, the disclaimer defined in point 1.1.7.
  • the compound binds to the kinase as measured by fluorescence polarization (FP), in particular to a Cys and/or His of said kinase.
  • FP fluorescence polarization
  • the Cys is a conservative Cys of said kinase protein, preferably of said kinase family or kinase subfamily.
  • the His is a conservative His of said kinase protein, preferably of said family or kinase subfamily.
  • the Cys is not a conservative Cys.
  • the His is not a conservative His.
  • the compound is measured as an inhibitor by any of the following assays: Z’LYTE Kinase Assay, ADP-Glo Assay, NanoBRET target engagement assay, 33 PanQinase kinase assay
  • Phosphorylation-Assisted Luciferase Complementation (PhALC) assay (Poti et al, 2023) orNanoBiT luciferase fragment-complementation assay (Dixon et al, 2016).
  • the compound is a compound as defined in point 2, or any of points 2 to 9, or preferably point 9, in particular point 9.1 or in particular point 9.2 or preferably any of points 10 to 15, preferably in point 16, e.g. 16.1.
  • the compound binds to a conservative Cys and/or a His residue of said protein with a reversible covalent bond.
  • the compound is as defined in point 16 or 18.
  • the compound binds to a conservative Cys residue of said protein with a reversible covalent bond.
  • the compound is RU60, RU67, RU68, RU75, RU76, RU128, RU81, RU169, RU188, RU83, RU100, RU101, RU102, RU103, RU189, RU115, RU140, RU141, RU214, RU215
  • the compound binds to a conservative His residue of said protein with a reversible covalent bond.
  • the compound is RU77.
  • the compound binds to either to a conservative Cys or a His residue of said protein with a reversible covalent bond.
  • the compound is RU187.
  • the compound of any of claims 1 to 16 for use in modulating a drug target protein associated with a disease is used as an inhibitor of a drug target protein associated with said disease.
  • the Michael-acceptor compound of the invention is used as a kinase inhibitor.
  • said disease is selected from the group consisting of an autoimmune disease, an inflammatory disease, a cardiovascular disease, a neurological disease or a disease of the nervous system, or a neoplasm or a cancer or malignancy, preferably a disease wherein kinase inhibition is required.
  • treatment involves prevention or avoiding or delaying the onset of a disease.
  • the one or more of the disclaimers defined in point 1.1 do not apply in the use of said compound as a kinase inhibitor.
  • disclaimer defined in point 1.1.1 the disclaimer defined in point 1.1.2, the disclaimer defined in point 1.1.3, the disclaimer defined in point 1.1.4, the disclaimer defined in point 1.1.5, the disclaimer defined in point 1.1.6, the disclaimer defined in point 1.1.7.
  • the kinase activated pathway is a MAPK kinase activated pathway, wherein preferably the disease is selected from the group consisting of an autoimmune disease, an inflammatory disease, a cardiovascular disease, a neurological disease or a disease of the nervous system, or a neoplasm or a cancer or malignancy, preferably an inflammatory disease, a neurological disease or a neoplasm or any variant thereof as defined herein.
  • the disease is selected from the group consisting of an autoimmune disease, an inflammatory disease, a cardiovascular disease, a neurological disease or a disease of the nervous system, or a neoplasm or a cancer or malignancy, preferably an inflammatory disease, a neurological disease or a neoplasm or any variant thereof as defined herein.
  • the kinase activated pathway is selected from an ERK1 kinase activated pathway, an ERK2 kinase activated pathway, an ERK5 kinase activated pathway, a JNK1 kinase activated pathway, a JNK2 kinase activated pathway, a JNK3 kinase activated pathway, a p38alpha kinase activated pathway, a p38beta kinase activated pathway, a p38gamma kinase activated pathway, p38delta kinase activated pathway.
  • the kinase activated pathway is selected from an ERK1 kinase activated pathway, an ERK2 kinase activated pathway, a JNK kinase activated pathway, a p38 kinase activated pathway or an ERK5 kinase activated pathway.
  • the kinase activated pathway is an ERK1/2, JNK and/or p38 kinase activated pathway, more preferably ERK2, JNK1, JNK2, JNK3, p38alpha kinase activated pathway.
  • the kinase activated pathway is an ERK2 kinase activated pathway, wherein preferably the disease is selected from the group consisting of an autoimmune disease, an inflammatory disease, a cardiovascular disease, a neurological disease or a disease of the nervous system, or a neoplasm or a cancer or malignancy, preferably an inflammatory disease, a neurological disease or a neoplasm or any variant thereof as defined herein.
  • the kinase activated pathway is a JNK1 kinase activated pathway, wherein preferably the disease is selected from the group consisting of an autoimmune disease, an inflammatory disease, a cardiovascular disease, a neurological disease or a disease of the nervous system, or a neoplasm or a cancer or malignancy, preferably an inflammatory disease, a neurological disease or a neoplasm or any variant thereof as defined herein.
  • the kinase activated pathway is a p38 kinase activated pathway, wherein preferably the disease is selected from the group consisting of an autoimmune disease, an inflammatory disease, a cardiovascular disease, a neurological disease or a disease of the nervous system, or a neoplasm or a cancer or malignancy, preferably an inflammatory disease, a neurological disease or a neoplasm or any variant thereof as defined herein.
  • the kinase activated pathway is an ERK1/2, JNK and/or p38 activated pathway, more preferably ERK2, JNK1, JNK2, JNK3, p38alfa activated pathway, wherein preferably the disease is selected from the group consisting of an autoimmune disease, an inflammatory disease, a cardiovascular disease, a neurological disease or a disease of the nervous system, or a neoplasm or a cancer or malignancy, preferably an inflammatory disease, a neurological disease or a neoplasm or any variant thereof as defined herein.
  • a neoplasm or a cancer or malignancy preferably a tumor, in particular a solid tumor, for example a breast, lung or colon tumor
  • a neurological disease or a disease of the nervous system preferably selected from the group consisting of neurological injuries, like acute brain injury, such as ischemic stroke (IS), intracerebral hemorrhage (ICH), subarachnoid hemorrhage (SAH), traumatic brain injury (TBI), spinal cord injury (SCI), epilepsy, etc; neurodegenerative diseases, such as Huntington’s disease (HD), Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), etc; neurodevelopment diseases, such as autism spectrum disorder (ASD) and cerebral palsy, as well as others; infectious neurological diseases, such as meningitis and encephalitis; and neurological tumors,
  • type 2 diabetes mellitus and obesity associated with insulin resistance a neoplasm or a cancer or malignancy, preferably a tumor, in particular
  • an inflammatory disease preferably atopic dermatitis, arthritis, e.g. psoriatic arthritis, rheumatoid arthritis, Crohn disease, ulcerative colitis, atherosclerosis, multiple sclerosis (MS), chronic obstructive pulmonary disease (COPD) and asthma.
  • arthritis e.g. psoriatic arthritis, rheumatoid arthritis, Crohn disease, ulcerative colitis, atherosclerosis, multiple sclerosis (MS), chronic obstructive pulmonary disease (COPD) and asthma.
  • MS multiple sclerosis
  • COPD chronic obstructive pulmonary disease
  • the disease is a disease related to impaired cell cycle, in particular selected from neoplasm and a neurological disease which is a cell cycle impairment disease or an aberrant cell cycle disease.
  • the kinase activated pathway is a JNK kinase activated pathway and the disease is selected from stomach cancer, oral squamous carcinoma, lung adenocarcinoma, cholangiocarcinoma, colon carcinoma, pancreatic cancer, glioblastoma, colon cancer, leukemia.
  • the kinase activated pathway is a p38 kinase activated pathway and the disease is selected from multiple myeloma, leukemia, oral epidermoid carcinoma, cervical cancer, melanoma, non-small cell lung cancer, ovarian cancer, glioma, myeloma, breast cancer, lung adenocarcinoma, colorectal, sarcoma, NSCLC, renal cancer, pancreatic cancer, and ovarian cancer.
  • the disease is selected from multiple myeloma, leukemia, oral epidermoid carcinoma, cervical cancer, melanoma, non-small cell lung cancer, ovarian cancer, glioma, myeloma, breast cancer, lung adenocarcinoma, colorectal, sarcoma, NSCLC, renal cancer, pancreatic cancer, and ovarian cancer.
  • the kinase activated pathway is an ERK1/2 kinase activated pathway and the disease is colorectal cancer.
  • the kinase activated pathway is an ERK5 kinase activated pathway and the disease is selected from lung cancer, cervical cancer, acute myeloid leukemia, pancreatic cancer, hepatocellular carcinoma, colon cancer.
  • kinase inhibitor is an inhibitor of a kinase of the family of mitogen-activated protein kinases (MAP kinases or MAPK), preferably the kinase is selected from extracellular regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 kinase, and extracellular regulated kinase 5 (ERK5), preferably from ERK2, JNK1, p38.
  • ERK1/2 extracellular regulated kinases 1/2
  • JNK c-Jun N-terminal kinase
  • p38 extracellular regulated kinase 5
  • ERK5 extracellular regulated kinase 5
  • the compound for use according to the invention binds to the MAPK D-groove.
  • the compound binds to a Cys or His residue of said protein which is conservative in MAP kinases, preferably in the MAPK D-groove, and binds with a reversible covalent bond.
  • the compound is as defined in point 16, 17 or 18.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the compound as defined in point 2, or any of points 2 to 9, or preferably point 9, in particular point 9.1 or in particular point 9.2 or preferably any of points 10 to 15, and a pharmaceutically acceptable excipient or carrier.
  • the invention also relates to a method of treatment wherein a pharmaceutical composition as defined in point 24 is administered to a subject in need thereof.
  • the compound is administered in sufficient dose by any appropriate means preferably as defined herein.
  • the subject is suffering in or jeopardized by a disease as defined in any of points 20 to 23.
  • Figure 1 shows the location of the MAPK D-groove cysteine (ERK2: Cysl61, p38a: Cysl62, and JNK1: Cysl63).
  • the panels show the crystallographic models of ERK2, p38a, and JNK1 bound to D-peptides (shown in black).
  • the sidechain atoms of the cysteines are shown with spheres.
  • the panels below show examples of the competitive fluorescence polarization-based protein-peptide assay for ERK2, p38a, and JNK1.
  • Panels on left show the direct titration curve using a fluorescently labelled peptide known to bind to the MAPK D-groove: a fixed amount of the labelled peptide was titrated with increasing amounts of human MAPKs (ERK2, p38oc, JNK1; wt: wild-type) and fluorescence polarization (FP) in arbitrary units was monitored. This gives the dissociation constant (Ka) for the binding of the labelled peptide with the respective MAPK. Panels on the right show the competitive binding curves with two compounds. (The MAPK concentration was chosen to start out with -50-90% complex formation with the labelled peptide; no competitor added; FB: fraction bound: -0.5-0.9).
  • the concentration of the small molecule was increased, and the inhibitory binding constant was determined (Ki app ). This is shown for two compounds (RU 1 and RU46). In this competitive binding setup, the fluorescent polarization signal will drop as the fluorescently labeled peptide is competed off from the MAPK docking groove.
  • Figure 2A-B show the structure -activity relationship (SAR) analysis of cyclohexenone containing compounds binding to ERK2.
  • Figure 2A shows the summary of competitive fluorescence polarization-based protein-peptide assays. The numbering of the carbon atoms of the cyclohexenone ring (1-6), the R’2 or R’4 substituent positions are indicated on the RU43 panel in the middle.
  • Figure 2B shows that mutating Cysl61 to alanine greatly decreases the binding affinity of RU64. Panels on the left show the direct binding titration with the proteins (wt or C161 A) and the reporter D-peptide (fluorescently labeled), panels on the right show the results of the binding competition experiments.
  • Figure 3A-C show the reversibility of ERK2 -cyclohexenone adduct formation.
  • Figure 3B shows real-time monitoring of ERK2-RU68 binding.
  • ERK2 was covalently linked to the SPR chip surface (CM-5) by amine-coupling.
  • RU68 was injected over this chip surface in 50 pM concentration for 10 minutes, then dissociation was followed for 20 minutes. Under these conditions the expected RUmax is -45. Notice the complete dissociation of the small molecule by the end of the experiment, which is consistent with reversible binding.
  • the panel shows the double -referenced binding curve using buffer injection correction against the experimental surface as well as sample injection correction against a control surface.
  • Figure 3C shows the intact mass before (left) and after dialysis (right) for ERK2-RU66, -RU81, and RU77.
  • Figure 4 shows the structural analysis of the ERK2-RU67 complex by X-ray crystallography.
  • the lower panel on the left displays the Fo-Fc omit map shown at 2o for the cysteine-RU67 covalent adduct.
  • ERK2 is shown in cartoon; the Cysl61-RU167 adduct and the main chain of 159-163 are shown in sticks.
  • Panels on the right show the ERK2-RU67 crystallographic model.
  • the docking groove is shown in surface representation and the Cysl61-small molecule adduct with sticks.
  • the lower panel also shows a D-peptide (pepMNKl, in gray) from the ERK2-pepMNKl (PDB ID: 2Y9Q) overlayed, highlighting that the small molecule’s cyclohexenone ring and the tert-butyl group bind to small hydrophobic pockets (q>A and q>B, respectively), while the bulky benzyl group faces towards q>B.
  • D-peptide pepMNKl, in gray
  • Figure 5A-B shows the comparison of different MAPK D-groove (ERK2 Cysl61) and JNK1 (Cysll6) targeting composite inhibitors.
  • Figure 5 A shows the location of the two different cysteines: the D-groove cysteine found in all three MAPKs (ERK, p38, and JNK) and the unique cysteine (Cysll6) in JNK1 located at its ATP-pockct/substratc binding pocket area.
  • the surface presentation of the respective region around the cysteines are shown in the lower panels from crystal structures (PDB ID: 2ERK, 5UOJ, 3V6S, and 2XRW).
  • FIG. 5B shows the measured apparent binding inhibitory values (Ki app ) of composite inhibitors comprised of two different directing groups (IN-8 scaffold for JNK1 Cysll6 or tert-butyl group for ERK2 Cysl61) and the same cyclohexenone warhead design connected via an amide or an ester.
  • the upper panels for JNK1 show the results of the NanoBRET target engagement assay (Reaction Biology Corp, USA). Duplicate measurements, error bars show SD.
  • Lower panels for ERK2 show the results of in vitro fluorescence polarization (FP) based binding assays. Triplicate measurements, error bars show SD.
  • Figure 6A-D show the extension of the cyclohexenone scaffold towards the CD-groove by click chemistry.
  • Figure 6A shows the crystal structure of the ERK2-pepMNKl crystallographic complex (PDB ID: 2Y9Q; top) and the model of the in silico docked (hypothetical) RU83-methyl-triazole molecule (bottom).
  • PDB ID: 2Y9Q crystallographic complex
  • bottom the model of the in silico docked (hypothetical) RU83-methyl-triazole molecule
  • the arrow shows the position of proline at q>L, whole the arrow on the lower panel shows the adjacent CD-groove.
  • Figure 6B shows how the CD-groove binding C-terminal half of the MNK1 peptide with N-terminal azide (N 3 -pepMNKl_C) was connected to RU83 containing an alkyne group at R’4 by copper-catalyzed azide/alkyne cycloaddition ('Bu: tert-butyl group).
  • FIG. 6C shows the summary of competition binding experiments: MAPK binding affinity (Ki) of the RU83-click- pepMNKl C chimera compared to RU83, N 3 -pepMNKl_C, and pepMNKl was measured using a fluorescence polarization-based assay, shown for two examples, with p38a and RU83 or RU83-click-pepMNKl_C (highlighted in bold font in the table) on the panels to the right; FB : fraction bound; * : competition is incomplete, Ki app is only estimated.
  • Figure 6D shows the results of SPR experiments with a p38a sensor chip.
  • Figure 7A-C show the extension of the cyclohexenone warhead scaffold towards the ATP binding pocket.
  • Figure 7A shows the structural model of JNK1 with a RU compound (RU60) bound in the D-groove and IN-8 in the ATP binding pocket.
  • Figure 7B shows the results of equilibrium binding SPR experiments in the presence of 1 mM GSH.
  • Figure 7C shows the kinetics of binding for IN-8 and RU128 injected over the JNK1 surface in a concentration corresponding to their equilibrium binding affinity (K D ). (Injection starts at time 0 and lasts for 5 minutes.)
  • Figure 8A-B show the cyclohexenone scaffold as a D-groove cysteine anchor to obtain MAPK-specific inhibitors in the presence of 10 mM GSH in vitro.
  • Figure 8A shows how the different stereoisomers of a RU compound at C4 (e.g., RU83 or RU188) could be used to direct additional moieties towards the CD-groove or to the ATP binding pocket via click chemistry.
  • the structure shows a model of the ERK2-RU60-ATP complex with pepMNKI C.
  • Figure 8B shows the summary of MAPK IC50 data determined by the PhALC assay (D-SENSOR, MEF2A) in the presence of 10 mM GSH.
  • the panels below show two examples of the PhALC measurements with JNK1 and RU 188 or RU 128, highlighted in bold font in the table. (Error bars show the SD of three independent experiments.)
  • Figure 9A-C show the characterization of different compounds in cell-based tests.
  • Figure 9A shows the characterization of RU83 and RU188 extended with a ⁇ ', ⁇ -diincthy lanilinc moiety (RU 103 and RU189) and tested in HEK293T using the NanoBiT protein-protein interaction (PPI) assay.
  • ERK2 binding partners MKK1, RSK1, and MKP3
  • MKK1, RSK1, and MKP3 interact via the MAPK D-groove (“-“ indicates no inhibitor was added).
  • the NanoBiT PPI assay detects higher level of binary protein binding interference in the presence of RU103 compared to its enantiomer (+RU103 vs +RU189).
  • NanoBiT luciferase fragment-complementation assay was done in live cells using 10 pM inhibitor concentration and 1.5 -hour compound pre-treatment before the measurements.
  • Figure 9B shows the effects of RU103 and RU189 on EGF-stimulated HeLa cells.
  • the cell line contains an AP-1 promoter+luciferase transgene cassette stably integrated into the genome.
  • Cells were unstimulated (-) or stimulated by Phorbol 12-myristate 13-acetate (+ PMA). Different inhibitors were added together with 10 nM PMA and the measurements were carried out after 6 hours. PMA-induced luciferase reporter gene transcription was monitored by luminescence measurements.
  • first panel inhibitors RU77, RU103 or RU189
  • were co -administered with PMA and were used in 1, 3, or 10 pM concentrations. (Error bars show standard deviation, N 3).
  • FIG. 10A-B show the structural comparisons of the ERK2 covalent adducts with different cyclohexenone containing compounds.
  • Figure 11 A shows the crystallographic complexes ERK2-RU67 or -RU68, ERK2-RU75 or RU76, and ERK2-RU60 or RU187.
  • 4 S or 4 R refer to the absolute configuration at C4; this is also shown with an arrowhead in the first panel, as well as the new asymmetric centers forming upon adduct formation at C2 and C3).
  • RU67/RU68, RU75/RU76, and RU60/RU187 are enantiomeric pairs.
  • the cysteine adduct in the ERK2-RU75 complex has two alternative C-S covalent bond conformations.
  • the ERK2 D-groove is shown with surface representation from the same view for RU67/RU68, RU75/RU76, and RU60 (position of q>B in the D-groove is shown to show the orientation and Cysl61 is pointed at with an arrowhead).
  • the crystal structure of the ERK2-RU187 complex is shown from a different angle.
  • This complex has a cysteine as well as a histidine adduct (and the D-groove is shown in surface representation apart from the amino acid adducts, which are shown in sticks).
  • one molecule can form only one of the two adducts, and the final structural model fitted best with the crystallographic data if the two alternative adducts were equally present.
  • Figure 10B shows the Fo-Fc omit maps contoured at 2o around Cysl61 and Hisl25 from the ERK2-RU60 and ERK2-RU187 crystal structures.
  • the red sphere in the ERK2-RU187 complex shows a water molecule H-bonded with the Hisl25- RU187 adduct and with the protein backbone (Serl22).
  • Figure 11A-B show the crystal structure of the ERK2-RU77 complex with the histidine adduct and the biochemical validation of histidine-adduct formation in the MAPK D-groove.
  • Figure 11 A shows the crystal structure of the ERK2- RU77 complex with the Fo-Fc omit map shown at 2o for the histidine-small molecule covalent adduct.
  • the panel on right shows the MAPK docking groove in surface representation and highlights the position of the different hydrophobic pockets (q>A, (
  • Figure 11B shows the results of the NanoBiT luciferase fragment-complementation assay.
  • the assay was done in live HEK293T cells using 10 pM inhibitor (RU77) concentration and 1.5-hour compound pretreatment before the measurements.
  • Figure 12 shows the crystal structure of composite inhibitors in complex with JNK1.
  • the BD837/BD838 warheads are enantiomers, and this structural comparison demonstrates that the C4 stereogenic center (pointed at with arrows for BD837/BD838 below) could be used to direct the carboxymethyl group towards different directions in the substrate binding pocket next to Cysll6.
  • Comparison of the JNK1-RU135-IN-8 and -BD837-IN-8 complexes shows that an additional methylene (highlighted with “*”) between the ATP pocket binding moiety and the warhead necessitates a dramatically different conformational solution to form the cysteine covalent adduct.
  • Lower panels display the Fo-Fc omit map for the cysteine-small molecule covalent adduct contoured at 1.5s.
  • Figure 13A-D show the comparative binding energetics, and “residence time” enhancement (i.e. k O ff decrease) due to reversible covalent bond formation for different Cysll6 targeting JNK inhibitors.
  • Figure 13 A shows the scheme of the 2-step reversible kinetic binding model with the relevant kinetic binding constants for noncovalent binding via the IN -8 ATP-binding moiety (kl and k2) as well as for warhead mediated covalent bond formation (k3 and k4) and the structure of the examined compounds.
  • kl and k2 the relevant kinetic binding constants for noncovalent binding via the IN -8 ATP-binding moiety
  • warhead mediated covalent bond formation k3 and k4
  • Figure 13B shows the SPR kinetic binding curves of two selected inhibitors (RU155-IN-8 and BD837-IN-8) as examples for the full analysis that was carried out for all the compounds shown on panel A (see Table 8).
  • the experimental kinetic binding plots collected at three different analyte concentration, chosen according to the compounds ’s K D are shown in black and the calculated plots (shown in gray) are based on the 2-step reversible kinetic binding scheme with the final kl, k2, k3 and k4 values.
  • Figure 13C shows the kinetic binding plots for JNK-IN-8 on the JNKl(Cysll6Ser) mutant surface injected at 100, 300, 1000 n concentrations (K D ⁇ 200-300 nM) as well as the response curves on the wildtype JNK1 surface (WT) at three different concentrations.
  • the analysis on the JNKl(Cysll6Ser) mutant surface gives the value of kl and k2 based on a one-site noncovalent binding model; these values were independently determined for each inhibitor this way (see Table 8).
  • Figure 13D shows binding energy contribution comparisons due to covalent bond formation through the respective warhead (AAG) and the comparison of off-rates (k2: noncovalent in gray vs k4: covalent in black).
  • AAG warhead
  • off-rates k2: noncovalent in gray
  • k4 covalent in black
  • JNK1-RU135-IN-8 covalent bond has ⁇ 100-fold increased off-rate compared to the more optimal conformation observed with the BD837 warhead.
  • a substantial gain in binding energetics as well as in on-site “residence” could be achieved by electronically tuning this suboptimal warhead by an ester instead of an amide at C2 (compare RU137-IN-8 with RU135-IN-8, respectively).
  • residence of the inhibitor is also affected by further sterical crowding at C4 (RU210 vs BD837) or by decreasing the flexibility of the cyclohexenone ring with the C4-C6 bridge (RU212 vs BD837).
  • k4 0 (and thus only k3 was fit)
  • k3 and k4 were both numerically fit to the experimental kinetic curves obtained on the JNK1 WT surface.
  • Figure 14A-B show the results of a stability assay in a nucleophile rich environment, in the presence of 10 mM GSH.
  • Figure 14A shows the inhibitory potential of JNK-IN-8 and BD837-IN-8 after pre-incubating the compounds for different amounts of time in 10 mM GSH before adding them into the PhALC assay.
  • Figure 14B shows the electrospray mass spectrometry characterization of JNK-IN-8 and BD837-IN-8 incubated in 10 mM GSH.
  • GSH-adduct formation is apparent in the different elution profile for JNK-IN-8 incubated with GSH for 18 hours vs no incubation (0 hr) as well as in the ion-chromatograms (RT: retention time; M + , M 2 7 M 3+ indicate the mass of differently charged molecule ions).
  • BD837-IN-8 stays intact and does not form an irreversible GSH adduct.
  • Figure 15A-E show the effects of JNK-IN-8 and BD837-IN-8 in cells on c-Jun phosphorylation.
  • Figure 15A shows the impact of the inhibitors on sorbitol stimulated JNK and c-Jun phosphorylation in HEK293T cells.
  • Cells were stimulated with sorbitol for 15 minutes which gives robust JNK activation. indicates no treatment with inhibitors and “+DMSO” indicates treatment with DMSO used in the same amount (0.1 %) as an organic solvent for the inhibitors.
  • Inhibitors were added 2 hours before stimulation. Note the changed electrophoretic mobility of JNK in the presence of JNK-IN-8, indicating irreversible JNK- JNK-IN-8 binding (compare lane 4-5 with the other lanes).
  • FIG. 15B shows the effects of JNK-IN-8 and BD837-IN-8 on c-Jun phosphorylation in an engineered neuroblastoma cell line (SH-SY5Y MKK7 ACT).
  • the phosphorylation of c-Jun was determined by using quantitative western blots (WB) using a phospo-c-Jun specific antibody (p-c-Jun).
  • Tubulin antibody was used as the load control and the p-c-Jun signal was divided by the tubulin signal, and the latter was normalized to p-c-Jun/tubulin measured in cells that were not treated by any inhibitor.
  • the lower panel shows a more detailed example of this type of analysis with BD838-IN-8.
  • Control cells were not treated with doxocycline (DOX) or inhibitor but only with 1% DMSO.
  • DOX doxocycline
  • Figure 15E show the results of short-term effects of JNK inhibitors in SH-SY5Y MKK7 ACT cells on c-Jun phosphorylation upon doxycycline (DOX) treatment for 2 or 4 hours in the absence (D2hr or D4hr, respectively) or in the presence of 0.7 pM DB837-IN-8 or JNK-IN-8.
  • Error bars indicate the SD calculated based on three independent experiments; p-values were calculated with a two-sided, paired t-test; “M” indicates the lanes with molecular weight markers.
  • Figure 16A-B show the effects of JNK inhibitors on cell viability and AP-1 promoter driven transcription.
  • Figure 16A shows the effect of inhibitors on JNK mediated cell death in SH-SY5Y MKK7 ACT cells. Endogenous JNK activation was initiated artificially by the addition of 2 pg/mL doxycycline (DOX) to engineered SH-SY5Y neuroblastoma cell line (in which the expression of an active MLK3-MKK7 chimera is controlled via the DOX dependent Tet-ON system).
  • DOX doxycycline
  • Cell viability was measured by monitoring the reducing power of living cells. The latter is a cell health indicator and can be monitored by fluorescence intensity measurements after addition of a resazurin-based solution (PrestoBlue).
  • the panel below shows the results of an experiment where cells were uninduced (Control) or induced doxycycline (+DOX) and treated with no inhibitor (DMSO) or with different inhibitors in 1 pM concentration. Cell viability was measured 72 hours after doxycycline and inhibitor co -administration. Notice that doxycycline treatment reduces cell viability but cells are well -protected by composite inhibitors containing covalent warheads (JNK-IN-8, BD837-IN-8, or BD838-IN-8).
  • the panel to the right shows the results of another experiment focusing on RU159_isoPHEN and the comparison of its effect to that of IN-8 and JNK-IN-8 (1 pM inhibitor concentration as in the other experiment but more cell death was initiated with doxycycline).
  • Figure 16B shows the effect of inhibitors on JNK mediated AP-1 transcription factor promoter activity.
  • Figure 17A-C show the specificity of BD837-IN-8.
  • Figure 17A shows the specificity of BD837-IN-8 on JNK1 in comparison to ERK2 and p38a in the PhALC assay. (Error bars show SD based on three experiments).
  • Figure 17B shows that JNK inhibitors do not affect the p38 MAPK pathway.
  • HEK293T MKK6EE cells (allowing p38-specific activation by doxycycline inducible expression of the constitutively active upstream activator kinase, MKK6EE, with a FLAG-tag) were stimulated for the indicated time by doxycycline (DOX) and cells were simultaneously treated with SB202190 (1 pM), a known p38-specific ATP -competitive inhibitor, or with different JNK inhibitors (3 pM).
  • the upper panel shows the western blot results with a phospho-MK2 specific antibody
  • the lower panel shows the western blot results using three antibodies (anti -tubulin - as the load control, anti-pp-p38 - recognizing the activated form of p38 kinases, and anti -FLAG - monitoring the expression level of MKK6EE-FLAG).
  • MK2 also known as MAPKAPK2
  • Figure 17C shows that BD837-IN-8 does not interfere with docking peptide (EvJIPl) binding.
  • a fluorescence polarization-based protein-peptide binding assay was used to detect the binding of the fluorescently labeled EvJIPl peptide in the JNK docking groove harboring Cysl63 (FB: fraction bound).
  • the panel on the left shows how the unlabeled EvJIPl peptide competes the labeled peptide off, and the panel on the right shows that the inhibitor does not interfere with docking motif binding, and thus demonstrating that this inhibitor does not target Cysl63 located in the JNK docking groove.
  • Figure 18 shows the Specificity of BD837-IN-8 in the human kinome panel.
  • Results of the Wild Type Kinase Panel (Reaction Biology Corp, USA; 340 human kinases) with BD837-IN-8 used at 1 pM concentration. Inhibition of a specific kinase is depicted with a circle on the human kinome tree, where circle size correlates with the amount of inhibition (and kinases are grouped based on sequence similarity into 7 kinase groups).
  • the panels below show the structural models of LIMK1 (PDB ID: 3S95) and TNK1 (homology model created by AlphaFold 2) superimposed with the JNK1-BD837-IN-8 crystallographic model. LIMK1 and TNK1 are the only two human kinases whose activity were inhibited more than 50% (see Table 10).
  • Figure 19A-B show JNK isoform specificity of cyclohexenone warhead containing inhibitors.
  • Figure 19A shows the crystal structure of the JNK1-BD837-IN-8 complex highlighting residues corresponding to exon 6 (shown with stick representation). This short region varies among JNK isoforms, and it forms the base of the substrate binding cleft next to the active site (D151). The three residues (GGV) displaying the greatest variation among the examined JNK isoforms are shown with spheres on the structural panel.
  • Figure 20A-C shows the effects of JNK PROTACs (RU219, RU231, RU232) on the level of JNK1 in HeLa cells.
  • Figure 20A shows the structure of three PROTACs with the same BD837-IN-8 based JNK specificity element linked to two different ubiquitin ligase binding (VHL or CRBN) moieities using two different linkers.
  • Figure 20B shows western blots results of three parallel experiments (1,2,3). The levels of JNK1 and p38oc were monitored using JNK1- or p38oc- specific antibodies and a tubulin-specific antibody was used as the load control (TUB).
  • TAB load control
  • FIG. 20C shows the quantitative summary of normalized JNK1 level changes or the p38oc/TUB WB signal ratio.
  • the JNK1 levels were normalized to the JNK1/TUB WB signal ratio of the “Conti”. Error bars show SD based on three independent experiments (*: p ⁇ 0.05; two-sided, unpaired t-test).
  • Figure 21 shows the results of cell proliferation experiments with different human cell lines with different Ras GTPases (A549 with RaS_G12S mutant, H1792 with Ras_G12C mutant, or LCL-103H wild-type with no mutation at position 12).
  • halo or halogen means fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine, even more preferably fluorine or chlorine.
  • alkyl alone or in combinations means a straight or branched -chain saturated hydrocarbon group containing from 1 to 6, preferably 1 to 5 carbon atom(s) (i.e. “Ci-e” or “C1.5” alkyl groups), such as methyl, ethyl, propyl, isopropyl, butyl, sec -butyl, tert-butyl and pentyl.
  • this phrase can relate to alkyl groups containing from 1 to 4, or 1 to 3, or 1 to 2 carbon atom(s) (i.e. “C1.4” or “C1.3” or “C1.2” alkyl groups), where the methyl or ethyl is a preferred embodiment.
  • alkenyl alone or in combinations means a straight or branched -chain hydrocarbon group containing from 2 to 6 carbon atoms or 2 to 5 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (i.e. “C2-6” or “C2-5” alkenyl groups).
  • this phrase can relate to alkenyl groups containing from 2 to 4, or 2 to 3 (i.e. “C2-4” or “C2-3” alkenyl groups).
  • Examples are ethenyl, 1 -propenyl, 2-propenyl, 1-butenyl, 2- butenyl, butadienyl, pentenyl, pentadienyl, hexenyl, and the like.
  • alkynyl alone or in combinations means a straight or branched-chain hydrocarbon group containing from 2 to 6 carbon atoms or 2 to 5 carbon atoms, one or more carbon-carbon triple bonds, and optionally one or more double bonds (i.e. “C2-6” or “C2-5” alkynyl groups).
  • this phrase can relate to alkynyl groups containing from 2 to 4, or 2 to 3 (i.e. “C2-4” or “C2-3” alkynyl groups). Examples are ethynyl, 1-propynyl, 2-propynyl, 1- butynyl, 2-butynyl, pentynyl, hexynyl, and the like.
  • haloalkyl means an alkyl group as defined above where one or more of the hydrogen atoms are substituted with halogen atoms.
  • alkyl, alkenyl and alkynyl are unsubstituted.
  • alkyl, alkenyl, and alkynyl are independently substituted with one or more substituents selected from the group consisting of -CN, -NO2, -NH 2 , -OH, - SH, -CO2H, -CHO.
  • aralkyl refers to an alkyl group substituted with an aryl group.
  • alkoxy refers to an alkyl group, as defined above, attached to the rest of the molecule through an oxygen atom.
  • amino means an optionally substituted -NH 2 group.
  • amino is substituted with one or two alkyl, preferably methyl or ethyl.
  • hydroxyl means an -OH group.
  • carbocyclyl alone or in combinations means a saturated or partially unsaturated cyclic hydrocarbon group having from 3 to 10 ring carbon atoms ("C3-10 cycloalkyl”) and zero heteroatoms in the non-aromatic ring system. In special cases, this phrase can relate to carbocyclyl groups containing from 3 to 8, or 3 to 6 (i.e. “C3-8” or “C3-6” cycloalkyl groups).
  • Non-limiting examples are cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptenyl, cycloheptadienyl, cycloheptatrienyl, cyclooctyl, cyclooctenyl, bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl, octahydro -I //-indcnyl.
  • carbocyclyl group is either monocyclic or contains a fused, bridged, or spiro ring system such as a bicyclic system.
  • Carbocyclyl also includes ring systems wherein the carbocyclic ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclic ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system.
  • Preferred carbocyclic groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
  • heterocyclyl alone or in combinations means a saturated or partially unsaturated non-aromatic ring system having 4 to 9 ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3-10 membered heterocyclyl”).
  • this phrase can relate to heterocyclyl groups having 3-8 ring forming atoms ("3-8 membered heterocyclyl”) or 4-6 ring forming atoms ("4-6 membered heterocyclyl”).
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • a heterocyclyl group can either be monocyclic ("monocyclic heterocyclyl") or a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic heterocyclyl”).
  • Non-limiting examples are azirdinyl, oxiranyl, thiorenyl, azetidinyl, oxetanyl, thietanyl, tetrahydrofuranyl, dihydroluranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, pyrrolyl-2,5- dione, dioxolanyl, oxasulfuranyl, disulfuranyl, oxazolidin-2-one, triazolinyl, oxadiazolinyl, thiadiazolinyl, piperidinyl, tetrahydropyranyl, dihydropyridiny
  • Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heterocyclyl also includes ring systems wherein the heterocyclic ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclic or heterocyclic ring, or ring systems wherein the heterocyclic ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclic ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclic ring system.
  • Preferred heterocyclyl groups are azetidine, oxetane, pyrrolidine, tetrahydrofuran, piperidine, pyran, morpholine, oxazine, dioxane.
  • carbocyclyl and heterocyclyl are unsubstituted.
  • carbocyclyl and heterocyclyl are independently substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkoxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted amino.
  • aryl alone or in combinations means an aromatic monocyclic or condensed double ring system comprising 6 to 10 carbon atoms.
  • suitable aryl groups include phenyl, and naphthyl, where phenyl is a preferred embodiment.
  • isosteres means groups with similar physical or chemical properties which produce broadly similar biological properties in the same chemical compound.
  • Phenyl isosteres are for example pyridinyl, thiophenyl, cubane-l,4-diyl, bicyclo[2.2.2]-octane-l,4-diyl and bicyclo[l.l.l]-pentane-l,4-diyl, preferably bicy clo[ 1.1.1 ]-pentane- 1,4-diyl.
  • heteroaryl means a group derived from a monocyclic or condensed double ring system with 1 to 3 ring forming heteroatom(s) selected from the group of N, O and S and 3 to 9 ring forming carbon atoms ("5-10 membered heteroaryl”). In special cases, this phrase can relate to heteroaryl groups having 5-8 ring forming atoms ("5-8 membered heteroaryl") or 5-6 ring forming atoms ("5-6 membered heteroaryl”).
  • Heteroaryl includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclic or heterocyclic groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system.
  • Heteroaryl also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system.
  • Non-limiting examples are pyrrolyl, luranyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl tetrazinyl, azepinyl, oxepinyl, thiepinyl, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl,
  • heteroaryl groups that contain one or more heteroatoms
  • the point of attachment can be a carbon or heteroatom, as valency permits.
  • Preferred heteroaryl groups are imidazole, pyrrole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, fiiran, thiophene, pyridine.
  • aryl, phenyl isosteres and heteroaryl are unsubstituted.
  • aryl, phenyl isosteres and heteroaryl are independently substituted with one or more substituents selected from the group consisting of halogen, amino, alkoxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted amino.
  • Preferred substituents are halogen, Ci-4 or C1.3 alkyl, C1.4 or C1.3 haloalkyl, C2-4 or C2-3 alkenyl, C2-4 or C2-3 alkynyl, amino, (C1.4 or Ci-3)alkylamino, di(Ci-4 or Ci-3)alkylamino, (C1.4 or C1.3) alkoxy.
  • N-protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999. Preferred examples are acetyl, benzoyl, benzyl, BOC, FMOC, carbobenzyloxy, carbamate and tosyl group.
  • a “chemically reasonable radical” means in the context of the present invention a molecular fragment which is stable at room temperature and atmospheric pressure.
  • a “chemically reasonable radical” in compounds of the formula I preferably means that bonding individual members of the list does not result in a chain moiety containing more than 2 heteroatoms (i.e. moieties such as -O-O-O- are excluded).
  • “Frustration” refers to sterical crowding effects by substituents on a moiety around the reactive center, specifically on a cycloalkenone moiety as used herein, which can block the formation of adducts with hindered amino acid residues endowing higher site selectivity for the ligands and mitigating “off -target’ ’ binding. Additionally, the same steric effect can result in the formation of a non-stable Michael adduct and facilitates the elimination of Michael-donor amino acid residues.
  • a “subject” as used herein is an individual of an animal species, preferably a vertebrate, more preferably a mammalian or avian species, in particular a mammalian species, highly preferably the individual is a primate, a hominid or a human.
  • a “patient” is a subject who is or intended to be under medical or veterinarian observation, supervision, diagnosis or treatment.
  • a “treatment” refers to any process, action, application, therapy, or the like, wherein the subject or patient is under aid, in particular medical or veterinarian aid with the object of improving or maintaining the subjects’s or patient’s condition, either directly or indirectly. Improving the subjects ’s condition may include restoring or maintaining normal function of an organ or tissue, preferably at least partly restoring or maintaining health (medical or veterinarian treatment). Treatment typically refers to the administration of an effective amount of a compound or composition described herein. Treatment may relate to or include medical or veterinarian treatment and cosmetic treatment, in particular medical or veterinarian treatment.
  • Preventing or “prevention” of the development of a disease or condition refers to at least the reduction of likelihood of the risk of or susceptibility to acquiring a disease or disorder, or preferably causing at least one of the clinical symptoms of the disease or disorder not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease.
  • a “neoplasm” is a type of abnormal and excessive growth of tissue. (The process that occurs to form or produce a neoplasm is called “neoplasia”.) The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists in growing abnormally, even if the original trigger is removed.
  • a malignancy is a malignant neoplasm.
  • tumor is used to describe a neoplasm, which is an abnormally increased mass of tissue or population of cells.
  • Cancer as used herein is a malignant tumor, and relates to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body.
  • Constant amino acids preferably a cysteine or histidine refer to amino acids found in related proteins in related positions. They share common biochemical properties for selective covalent targeting by an electrophile.
  • a conservative amino acid (preferably cysteine or histidine) of a protein family is understood herein as an amino acid (a cysteine or histidine) present in a given related position in the majority or most of the members of the protein family or in a subgroup.
  • MAP kinases mitogen-activated protein kinases
  • MAPK mitogen-activated protein kinases
  • MAPK subfamilies like ERK1, ERK2, JNK, p38 kinases, ERK5 etc.
  • the related position is a position which is important for or has a role in the biochemical properties of the related proteins, e.g. throughout a given protein family or variant group of said protein family.
  • MAP kinases or MAPK are mitogen-activated protein kinases and the term are used interchangeably.
  • extracellular regulated kinase 1 ERK1
  • ERK1 extracellular regulated kinase 1
  • extracellular regulated kinase 2 ERK2
  • ERK2 extracellular regulated kinase 2
  • ERK2 kinase extracellular regulated kinase 2
  • extracellular regulated kinase 5 ERK5
  • ERK5 extracellular regulated kinase 5
  • JNK1 c-Jun N-terminal kinase 1
  • JNK1 JNK1
  • JNK2 c-Jun N-terminal kinase 2
  • JNK2 JNK2 kinase
  • JNK3 JNK3 kinase
  • the present invention provides pharmaceutical compositions comprising a compound of the present invention, e.g., a compound of Formula (I), and pharmaceutically acceptable salts thereof, as described herein, and optionally a pharmaceutically acceptable excipient.
  • a compound of the present invention e.g., a compound of Formula (I)
  • pharmaceutically acceptable salts thereof as described herein, and optionally a pharmaceutically acceptable excipient.
  • the compound of the present invention, or a pharmaceutically acceptable salt thereof is provided in an effective amount in the pharmaceutical composition.
  • the effective amount is a therapeutically effective amount.
  • the effective amount is a prophylactically effective amount.
  • compositions described herein can be prepared by any method known in the art of pharmacology.
  • preparatory methods include the steps of bringing the compound of the present invention (the "active ingredient") into association with a carrier and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single - or multi-dose unit.
  • compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • compositions used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition. These ingredients are well known in the state of the art.
  • the compounds and compositions of the invention can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • enteral e.g., oral
  • parenteral intravenous, intramuscular, intraarterial, intramedullary
  • intrathecal subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal
  • topical as by powders, ointments, creams, and/or drops
  • mucosal nasal, buccal
  • Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site.
  • intravenous administration e.g., systemic intravenous injection
  • regional administration via blood and/or lymph supply e.g., systemic intravenous injection
  • direct administration e.g., direct administration to an affected site.
  • the formulations appropriate for said administration routes are well known in the state of the art.
  • Dosage forms for injectable preparations for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol.
  • compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates of this invention with suitable nonirritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • suitable nonirritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier and/or (a) fillers or extenders (b) binders, (c) humectants, (d) disintegrating agents, (e) solution retarding agents, (f) absorption accelerators, (g) wetting agents, (h) absorbents, and (i) lubricants, and mixtures thereof.
  • the dosage form may comprise buffering agents.
  • Dosage forms for topical and/or transdermal administration of a compound of this invention may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches.
  • the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier and/or any needed preservatives and/or buffers as can be required.
  • the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body.
  • Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium.
  • the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.
  • the exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like. Determination of the amount to be administered is within the competence of the skilled practitioner and represents an aspect of the state of the art.
  • Compounds according to the present invention can be used alone or in combination with other agents, wherein at least one compound according to the present invention is administered simultaneously, consecutively or prior to the other treatment.
  • the present invention provides compounds for use in the prevention and treatment of various diseases, such as neurodegenerative diseases, e.g. Parkinson's disease, Alzheimer's disease, regeneration/repair after an injury to the central nervous system, e.g. axonal regeneration, metabolic disorders, e.g. diabetes, inflammatory diseases, cardiovascular diseases, e.g. stroke and hypertension, and proliferative diseases, e.g., cancer and benign neoplasms.
  • neurodegenerative diseases e.g. Parkinson's disease, Alzheimer's disease
  • Alzheimer's disease e.g. axonal regeneration
  • metabolic disorders e.g. diabetes
  • cardiovascular diseases e.g. stroke and hypertension
  • proliferative diseases e.g., cancer and benign neoplasms.
  • the disease is a disease associated with JNK activity, e.g., a disease associated with aberrant or unwanted JNK activity.
  • the disease results from increased JNK activity.
  • the compounds of the present invention, or the pharmaceutical compositions thereof are useful in the treatment of JNK-associated disease.
  • Inhibition of JNK1 is associated with the treatment of cancer, diabetes, and inflammatory diseases (e.g., inflammation).
  • Increased JNK1 activity is also associated with obesity, i.e., inhibition of JNK1 or mouse knockout has been found to increase insulin sensitivity (Hirosumi et al., 2002).
  • Inhibition of JNK3 is associated with the treatment of neurodegenerative diseases.
  • the disease is a disease associated with ERK activity, e.g., a disease associated with aberrant or unwanted ERK activity.
  • the compounds of the present invention, or the pharmaceutical compositions thereof are useful in the treatment of ERK -associated disease, particularly in cancer (Smalley et al, 2018).
  • the disease is a disease associated with p38 activity, e.g., a disease associated with aberrant or unwanted p38 activity.
  • the compounds of the present invention, or the pharmaceutical compositions thereof are useful in the treatment of p38-associated disease, particularly in inflammation, immune disorders or cancer (Gupta et al., 2015) (Canovas et al, 2021).
  • Trifluoroacetic acid (811 pL, 10.53 mmol, 3 eq.) was added to a solution of compound 4 (942 mg, 3.51 mmol, 1 eq.) in methylene chloride (20 mL) and then the mixture was stirred at 25°C. The reaction was monitored by TLC and when it was completed ( ⁇ 3 hours) the reaction mixture was evaporated. The remaining trifluoroacetic acid was removed by redissolving the mixture in toluene and evaporating to dryness at 50°C three times. Compound 5 was obtained as a yellow oil in quantitative yield (745.0 mg) and was used in the next step without further purification.
  • Aflame-dried vial was charged with an anhydrous toluene solution (3.5 mL) of RU41 (1.00 g, 3.40 mmol, 1 eq.) and triethylamine (940 pL, 6.79 mmol, 2 eq.) under a nitrogen atmosphere.
  • the reaction mixture was cooled to 0°C then TBDMSOTf (936 pL, 4.08 mmol, 1.2 eq.) was added dropwise, and it was stirred at 0°C.
  • the reaction was completed ( ⁇ 2 hours), the mixture was diluted with water and extracted with hexanes three times. The combined organic phases were washed two times with saturated NaHCCh solution and once with brine, dried over anhydrous NaiSO i. filtered and the solvent was removed under reduced pressure.
  • the silyl-enol ether intermediate was obtained as a colorless oil (1.307 g, 94%) and was used in the next step without further purification.
  • Trifluoroacetic acid (312 pL, 4.05 mmol, 3 eq.) was added to a solution of compound RU217/218 (400.0 mg, 1.35 mmol, 1 eq.) in methylene chloride (10.4 mL) and then the mixture was stirred at 25°C. The reaction was monitored by TLC and when it was completed ( ⁇ 3 hours) the reaction mixture was evaporated. The remaining trifluoroacetic acid was removed by redissolving the mixture in toluene and evaporating to dryness at 50°C three times. Compound 9 was obtained as a yellow oil in quantitative yield (324.0 mg) and was used in the next step without further purification.
  • Trifluoroacetic acid (158 pL, 2.05 mmol, 3 eq.) was added to a solution of compound RU73/77 (200.0 mg, 0.684 mmol, 1 eq.) in methylene chloride (4.0 mL) and then the mixture was stirred at 25°C. The reaction was monitored by TLC and when it was completed ( ⁇ 3 hours) the reaction mixture was evaporated. The remaining trifluoroacetic acid was removed by redissolving the mixture in toluene and evaporating to dryness at 50°C three times. Compound 10 was obtained as a yellow oil in quantitative yield (160.0 mg) and was used in the next step without further purification.
  • Compound 13 was prepared according to the literature procedures (Schultz et al. 1984) (Peelen et al., 2004) (Meyer et al. 2000).
  • reaction was stirred at 25°C for 16 h, over which time the solid dissolved.
  • a saturated aqueous NaHSCh solution was added until the purple color disappeared.
  • the reaction mixture was filtered through a pad of Celite which was washed two times with diethyl ether to remove the brown solid.
  • the filtrate was acidified with cone. HC1 until pH 1.
  • the reaction was washed with diethyl ether three times, and the combined extracts were dried over anhydrous NJBSO i. the solvent was evaporated under reduced pressure, affording compound 15 as a colorless oil without further purification (240.0 mg, 47%).
  • the IN-7, 8, 9, 10 scaffolds (20a-d) were synthesized according to the literature procedures (see in Qian et al, 2019).
  • CN-9 (77.3 mg, 0.504 mmol, 2 eq.) was dissolved in anhydrous dimethylformamide (1.0 mL) in a flame-dried vial under a nitrogen atmosphere, HBTU (191.3 mg, 0.504 mmol, 2 eq.) and A-methylmorpholine (111 pL, 1.01 mmol, 4.0 eq.) were added and the mixture was stirred at 25°C. After 30 minutes an anhydrous DMF solution (1.0 mL) of compound 20b (100 mg, 0.252 mmol, 1 eq.) was added and the reaction mixture was stirred at 25°C for 3 days.
  • 13 C (125 MHz, CDCh): 207.6, 181.5, 165.3, 162.6, 161.0, 159.2, 159.1, 151.2, 148.3, 138.1, 136.1, 134.9, 134.5, 134.3, 133.9, 130.6, 129.6, 125.8, 124.8, 123.6, 123.4, 123.3, 122.7, 118.8, 118.7, 108.0, 51.5, 39.1, 27.5, 18.4.
  • the reaction was monitored by HPLC-MS and when the starting material was consumed the reaction mixture was diluted with ethyl acetate, washed two times with a 5% citric acid solution, two times with a saturated aqueous NaiCOi solution, and two times with brine. The organic phase was dried over anhydrous NaiSOi. filtered, then evaporated under reduced pressure. The resulting crude product was purified by flash column chromatography on silica gel using hexanes:ethyl acetate as eluent, affording the Boc-protected amine as an off-white solid (24.9 mg, 40%).
  • Trifluoroacetic acid (159 pL, 2.06 mmol, 3 eq.) was added to a solution of compound RU83/RU188 (201 mg, 0.69 mmol, 1 eq.) in methylene chloride (5 mL) and then the mixture was stirred at 25°C. The reaction was monitored by TLC and when it was completed (3 h) the reaction mixture was evaporated. The remaining trifluoroacetic acid was removed by redissolving the mixture in toluene and evaporating to dryness at 50°C under reduced pressure which was repeated three times. Compound 5’ was obtained as a yellow oil in quantitative yield (162 mg) and was used in the next step without further purification.
  • Apressure vessel was charged with compound 49 (0.750 g, 3.19 mmol 1 eq.), (3-aminophenyl)boronic acid (0.655 g, 4.78 mmol, 1.5 eq.), Na 2 CO 3 (0.676 g, 6.38 mmol, 2 eq.), Pd(PPh 3 )4 (0.220 g, 0.19 mmol, 0.059 eq.), 9.6 mL dimethoxyethane and 4.8 mL water and the reaction mixture was stirred at 85°C for 18 hours under a N 2 atmosphere. After completion the reaction mixture was cooled to 25°C and water and ethyl acetate was added to it.
  • a pressure vessel was charged with a solution of compound 50 (0.400 g, 1.61 mmol, 1 eq.) in 8 mL methanol and 94 pL 98% acetic acid.
  • the atmosphere was changed to N 2 then palladium on carbon was added (0.171 g, 0.161 mmol, 0.1 eq.) and vessel was charged with 10 bar H 2 and the reaction mixture was stirred at 25°C for 24 hours. After completion the reaction mixture was filtered on a pad of Celite then evaporated.
  • the crude mixture was dissolved in methylene chloride then washed 2 times with a 10% NaOH solution. The organic phase was washed with water and brine, then it was dried over anhydrous Na 2 SO4, filtered and the solvent was evaporated under reduced pressure giving compound 51 as an orange oil (0.230g, 56%).
  • 2-azidoacetic acid (68.0 mg, 0.670 mmol, 1.8 eq.) was dissolved in anhydrous dimethylformamide (1.0 mL) in a flame- dried vial under a nitrogen atmosphere, HBTU (260.0 mg, 0.670 mmol, 1.8 eq.) and ⁇ '-mcthylmorpholinc (120 pL, 1.10 mmol, 3.0 eq.) were added and the mixture was stirred at 25°C.
  • anhydrous DMF solution (3.0 mL) of compound 19 (100.0 mg, 0.252 mmol, 1 eq.) was added and the reaction mixture was stirred at 25°C for 3 days.
  • the reaction was monitored by HPLC-MS and when the starting material was consumed the reaction mixture was diluted with ethyl acetate, washed two times with a 5% citric acid solution, two times with a saturated aqueous Na2CCh solution, and two times with brine.
  • the organic phase was dried over anhydrous NaiSOi. filtered, then evaporated under reduced pressure.
  • the azidoacetic acid was synthesized according to literature procedure (Schmitz et al, 2016).
  • the peptide was chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Arlington) with standard Fmoc/'Bu strategy. After solid phase peptide synthesis and subsequent cleavage from resin, the peptides were isolated by semi-preparative RP-HPLC using water with 0.1% formic acid as eluent A and acetonitrile with 0.1% formic acid as eluent B.
  • 70.42 (m), 70.39, 70.37, 70.35, 70.0, 69.3, 67.1, 59.0, 58.6, 57.7, 56.7, 50.3, 45.1, 43.1, 36.6, 36.2, 35.7, 35.1, 31.9, 26.4, 24.3, 18.4, 16.0.
  • Compound 54 was synthesized according to literature procedure from 6 -aminohexanoic acid (Ha et al., 2017).
  • Compound 56 was purchased from Advanced ChemBlocks Inc, USA.
  • Activated ERK2, p38a, and JNK1 for the biochemical assays were produced by co-expressing the MAPKs with constitutively active GST-tagged MAP2Ks in E. coli with bicistronic plasmids (GST-MKK1 3D/ERK2, GST- MKK6_EE/p38a, GST-MKK7 EE/JNK1/2). Double phosphorylation of the activation loop of MAPKs was confirmed by western blot analysis with anti-phospho MAPK-specific antibody and/or mass spectrometry.
  • MAPKs were expressed with an N-terminal His6-tag that was cleaved off by the TEV protease after purification with Ni-NTA affinity resin, and then samples were further purified using ion-exchange chromatography (on MonoQ column). Dephosphorylated MAPKs were produced with GST-tagged /.-phage phosphatase and purified similarly. Human JNK3 was expressed alone using a simpler bacterial expression vector, and its activated form was produced by incubating the protein with constitutively active version of MKK7 in vitro before kinase activity measurements. GST-MKK7EE-Hise was expressed in bacteria and double-affinity purified.
  • Biotinylated MAPKs for the SPR experiments were co-expressed with the BirA ligase in E. coli and purified as described above but this construct retained an N-terminal AviTag after TEV cleavage.
  • Peptides were chemically synthesized using solid phase peptide synthesis (on Rink Amid resin, PS3 peptide synthesizer, Protein Technologies) with Fmoc/'Bu strategy and purified by RP-HPLC using a Jupiter 300 A C18 column (Phenomenex). Quality of the peptides were checked by HPLC-MS (Shimadzu LCMS-2020) JNK-IN-8 was purchased from Selleck Chemicals, USA. Tert-Butyl acrylate was purchased from Sigma-Aldrich Chemicals, USA
  • FP Fluorescence polarization assay
  • the unlabeled competitor (peptide or small molecule) was added in increasing amounts and the FP signal was measured in a Cytation 3 (BioTek Instruments) fluorescence plate reader in 384-well plates (CORNING, Low Volume, Round Bottom, Non-Binding Surface) in 20 pL volume.
  • the K, for each competitor was determined by fitting the data to a competition binding equation. Titration experiments were carried out in triplicates, and the average FP signal was used for fitting the data in Origin 2018 (OriginLab, USA).
  • the standard weighted least squares method was used to determine the Ki estimate, with its error given by the corresponding diagonal element of the covariance matrix.
  • the Phosphorylation-Assisted Luciferase Complementation assay was developed to measure MAPK activity in vitro for fast and cost-effective identification/characterization of any compound blocking MAPK activity (Poti et al, 2023).
  • the principle of this assay is the following: the general MAPK phosphorylation target motif (S/TP) is positioned C-terminal from a MAPK binding D -motif and this SENSOR construct is fused with the small fragment of the luciferase enzyme.
  • the WW domain (Pint protein) binding specifically to the phosphorylated MAPK target motif is fused with the large fragment of the luciferase enzyme (Dixon et al., 2016) (Lu et al., 1999).
  • the RC Upon SENSOR phosphorylation the RC binds to the SENSOR and triggers the assembly of the luciferase enzyme which will produce photons as it turns over its substrate (coelenterazine).
  • Both constructs are produced with an N-terminal maltose binding protein (MBP) and a C-terminal histidine-tag for high-yield bacterial expression and for simple affinity -resin purification.
  • MBP N-terminal maltose binding protein
  • C-terminal histidine-tag for high-yield bacterial expression and for simple affinity -resin purification.
  • the two purified constructs (SENSOR and RC) were mixed with activated MAPKs, the reaction was started by injecting ATP into the reaction mix containing the luciferase enzyme substrate coelenterazine, and the luminescence signal was monitored in time.
  • the PhALC assay was used as a semi-high throughput, microplate compatible biochemical assay to obtain IC50 values of any compounds blocking MAPK activity.
  • RC and SENSOR were typically used in 1 pM concentrations with 1-10 nM double-phosphorylated MAPKs produced and purified as described earlier (Zeke et al., 2015).
  • the coelenterazine concentration was 200 pM and the reaction was started by adding 0.1-1 mM ATP.
  • the luminescence signal was monitored up to 30 minutes and the slope at the linear range (typically up to 5 minutes) was calculated based on linear regression.
  • the p38 and ERK D- SENSOR contained the CNK3 (LKKEKSAILDLYIPP) or MEF2A D-motif (SRKPDLRVVIPP) and the JNK D- SENSOR had the more JNK-specific pepPDE4B motif (GDGISRPTTLPLTTLP) (Garai et al., 2012) (Zeke et al., 2015).
  • SENSORs contained the same MAPK phosphorylation target sequence compatible with WW domain binding: VPRTPVS. This sequence motif was positioned C-terminal from the D-motif in D-SENSORs, separated by a flexible linker (HMGSGSSGGSSGSGSVD).
  • ERK2 was captured on a Biacore CM5 sensor chip by amine coupling using a Biacore S200 instrument (GE -Healthcare).
  • the SPR running buffer was the following: 1 X PBS supplemented with 0.05% Tween20, 0.2 mM TCEP, 5% DMSO.
  • the protein was expressed with an N-terminal Hisw-tag and an NTA sensor chip was used for capturing the protein, and the SPR running buffer was the following: 10 mM Tris pH 8.0, 300 mM NaCl, 0.2 mM TCEP, 0.05% Tween20.
  • the proteins were expressed biotinylated with an N-terminal AviTag, expressed in bacteria, purified, and immobilized on a Biacore CAP sensor chip, and the SPR running buffer was the following: 10 mM HEPES pH 7.4, 150 mN NaCl, 0.05% Tween20, 1 mM GSH, 1% DMSO. All measurements were done by using a Biacore S200 instrument (GE -Healthcare) at room temperature. The K D was determined based on RU values corresponding to different concentrations of the analyte using single-cycle setup with the standard Biacore method for CAP chip including double referencing. Sensorgrams were fit to a 1:1 binding model using the BiaEvaluation software (GE Healthcare).
  • the kinetic binding parameters (k O ff) were determined from sensorgrams obtained at an analyte concentration corresponding to the equilibrium binding constant (KD).
  • the SPR running buffer was the following: 1 mM HEPES, 150 mM NaCl, 0.05 % TWEEN, 1 mM GSH, 1% DMSO.
  • JNK1 measurements the protein was expressed biotinylated on an N-terminal AviTag, expressed in bacteria, purified, and immobilized on a Biacore CAP sensor chip.
  • the determination of kl, k2 (noncovalent k on and k O ff, respectively) and k3, k4 (covalent k on and k O ff, respectively) were determined the following way.
  • Inhibitors were injected over the JNKl(Cysll6Ser) mutant surface at three different concentrations (100 nM, 300 nM, and 1000 nM) and this kinetic data was globally fit with to 1 : 1 binding model using the BiaEvaluation software (GE Healthcare), which gave the kl and k2 value for each inhibitor.
  • the inhibitors were injected at three different concentrations (depending on their estimated K D determined earlier by independent equilibrium measurements) over the CAP sensor chip loaded with wild-type JNK1 and k3 and k4 values were determined by fitting the kinetic binding curves using the COPASI biochemical modeling software based on a 2 -step reversible binding scheme, where kl and k2 values were fixed (Hoops et al, 2006).
  • the molecular weights of MAPKs and their conjugates were identified using a Triple TOF 5600+ hybrid Quadrupole - TOF LC/MS/MS system (Sciex, Singapore, Woodlands) equipped with a DuoSpray lonSource coupled with a Perkin Elmer Series 200 micro LC system (Massachusetts, USA). Data acquisition and processing were performed using Analyst TF software version 1.7.1 (AB Sciex Instruments, CA, USA). Chromatographic separation was achieved by Thermo Beta Basic C8 (50 mm x 2,1mm, 3 pm, 150 A) HPLC column. Sample was eluted in gradient elution mode using solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in ACN).
  • the molecular weights of the conjugates of GSH were identified using a Triple TOF 5600+ hybrid Quadrupole-TOF LC/MS/MS system (Sciex, Singapore, Woodlands) equipped with a DuoSpray lonSource coupled with a Shimadzu Prominence LC20 UFLC (Shimadzu, Japan) system consisting of binary pump, an autosampler and a thermostated column compartment. Data acquisition and processing were performed using Analyst TF software version 1.7.1 (AB Sciex Instruments, CA, USA). Chromatographic separation was achieved on a Phenomenex Luna Omega PS C18 (50 mm x 2,1mm, 3 pm, 100 A) HPLC column.
  • Sample was eluted in gradient elution mode using solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in ACN).
  • the initial condition was 10% B followed by a linear gradient to 55 % B by 12 min, to 95 % B by 3 min, 15 to 17.5 min 95% B was retained; and from 17.5 to 18 min back to initial condition with 10 % eluent B and retained from 18 to 20 min.
  • Flow rate was set to 0.4 ml/min.
  • the column temperature was 40 °C and the injection volume was 5 pl.
  • UV-VIS spectrometer was used in 254 nm wavelength.
  • Nitrogen was used as the nebulizer gas (GS1), heater gas (GS2), and curtain gas with the optimum values set at 30, 30 and 35 (arbitrary units), respectively.
  • the source temperature was 350 °C and the spray voltage was set to 5500 V. Declustering potential value was set to 80 V.
  • Peak View SoftwareTM V.2.2 version 2.2, Sciex, Redwood City, CA, USA was used for deconvoluting the raw electrospray data to obtain the neutral molecular masses.
  • NanoBRET target engagement assay was carried out by Reaction Biology Corp (USA, Malvern). Briefly, HEK293 cells were transfected with 1 pg JNK1 and 9 pg transfection carrier DNA. The transfected cells were treated with compounds (starting at 1 or 100 pM, 10-dose with 3-fold dilution). JNK1 target engagement was measured by NanoBRET assay. The K-5 tracer concentration was 0.5 pM. Compound treatment time was 2 hours.
  • HEK293T cells were transfected with LgBiT and SrnBiT containing plasmids using Lipofectamin 3000 in Opti-MEM (Gibco).
  • cDNAs were sub-cloned into LgBiT and SrnBiT expression vectors: ERK2 and p38a were expressed as LgBiT fusions and partner proteins constructs had Smbit fusion tags.
  • HeLa cells were seeded into 48-well plates and were grown till 80 % confluence (typically 24 hrs) in 200 pl DMEM supplemented with 10 % FBS and were serum-starved (0 % FBS) for 16 hours before pretreatment with inhibitors (10 pM) for 1.5 hour.
  • Cells were stimulated by 100 ng/mL EGF for the indicated time, the media was removed, and cells were lysed in 70 pl IX SDS-PAGE loading buffer, 10 pl was loaded onto 4-20 % Mini-PROTEAN TGX Precast gels (Bio-Rad) 5-15 % Tris-glycine SDS-PAGE gels and gels were blotted to nitrocellulose membrane.
  • the DMSO control contained 0.05 % DMSO, an equal amount to inhibitor treated cells.
  • Western blot results were analyzed using Odyssey CLx imaging system (Li-Cor) and fluorescently labeled secondary antibodies: IRDye 680RD (Goat anti-mouse IgG; Li-cor #925-68070; 1: 10000) or IRDye 800CW (Goat anti-rabbit IgG, Li-Cor #926-32211; 1:5000).
  • the primary antibodies were the following: anti-p44/42 MAPK (ERK1/2) (L34F12) Mouse mAb (Cell Signaling #4696; 1:3000 dilution; referred to as ERK antibody), anti -pho spho-p44/42 MAPK (Thr202/Tyr204) Rabbit Ab (Cell Signaling #9101; 1:3000 dilution; referred to as ppERK), anti-phospho-p90RSK (S380) (D3H11) Rabbit mAb (Cell Signaling 11989; 1:2000 dilution; referred to as pRSK), anti-a-tubulin (Sigma #T6199; 1:10000 dilution, referred to as TUB).
  • 50,000 HEK293T cells were seeded into 24-well plates and were grown till confluence (typically 24 hrs) in 500 pl DMEM supplemented with 10 % FBS and were serum-starved (0% FBS) for 16 hours before sorbitol (0.35 M) treatment for 15 minutes.
  • the plate was put on ice and cells were resuspended, pelleted by centrifugation, and washed with ice cold PBS.
  • the cell pellet was lysed in 80 pl IX SDS-PAGE loading buffer, 10 pl was loaded onto 10 % Tris- glycine SDS-PAGE gels and gels were blotted to nitrocellulose membrane.
  • Inhibitors (1 pl dissolved in 50 % DMSO) were added 2 hours before sorbitol treatment.
  • SH-SY5Y MKK7 ACT cells allowing specific activation of JNK upon addition of an inducer, were handled similarly but JNK activation was induced by the addition of 3 pg/ml doxycycline.
  • IRDye 680RD Goat anti-mouse IgG; Li-cor #926-68070; 1 : 10000
  • IRDye 800CW Goat anti-rabbit IgG, Li-Cor #926-32211 ; 1 :5000.
  • the primary antibodies were the following: anti-phospho JNK (Cell Signaling #9251; 1:1000), anti-phospho-c-Jun(Ser73) (Cell Signaling #9164; 1: 1000), anti-phospho p38 (Cell Signaling #9215, 1:3000), anti-phospho MK2 (Cell Signaling #3007, 1:1000), anti-a- tubulin (Sigma #T6199; 1:10000), or anti-FLAG (Sigma #F1804; 1:10000).
  • Anti-a-tubulin antibody was used as the load control and the anti-FLAG antibody was used to monitor the expression of the transgene responsible for specific MAPK activation.
  • AP-1 Reporter HEK293 cells contains a firefly luciferase gene under the control of AP-1 responsive elements that are stably integrated into HEK293 cells.
  • 20,000 Reporter AP-1 - HEK293 cells were seeded into a 48-well plate in 200 pl DMEM (Gibco). After cells become adherent (confluency: 80 %) the inhibitors were added in opti-MEM (Gibco) and following 2 hours of incubation cells were stimulated with 6 ng/ml PMA solution for 6 hours. The luminescence signal was read out using a BioTek Cytation 3 microplate reader after adding freshly prepared Steadylite Plus solution (PerkinElmer) according to the manufacturer's protocol.
  • the SH-SY5Y (neuroblastoma) MKK7 ACT cell line was generated using lentiviral transduction with an MKK7(ACT)-MLK3 construct containing full-length human MKK7 fused to the kinase domain of human MLK3 (aa. 117-379).
  • the generated Tet-ON inducible stable cell line was evaluated by time dependent transgene induction and downstream target phosphorylation upon doxycycline treatment, as described earlier (Kirsch et al., 2020).
  • JNK inhibitor mediated c-Jun phosphorylation cells were stimulated with 2 pg/mL doxycycline, coadministered with different amounts of inhibitors (between 30 pM and 30 or 10 nM concentrations in three-fold dilutions with 7 or 8 data points), and samples were subjected to quantitative western blot (WB) analysis after 6 hours.
  • WB western blot
  • the standard weighted least squares method was used to determine the EC50 estimate, with its error given by the corresponding diagonal element of the covariance matrix.
  • the PrestoBlue Cell Viability Reagent (ThermoFisher, P50200) was used to assess cell viability of SH-SY5Y MKK7 ACT cells.
  • 3,000 SH-SY5Y MKK7 ACT cells were plated in 96-well plates in DMEM with 10% FBS. Next day the medium was changed for new media (DMEM with 0.1 % of FBS) and cells were incubated with inhibitors for 72 hours. The medium was then removed and PrestoBlue reagent was added to live cells, incubated for 45 minutes, then the fluorescence signal (ex: 560 nm, em: 600 nm) was measured in a plate reader. Linearity of the detection was checked in a parallel experiment where control cells were serially diluted, and the signal was measured according to the manufacturer's protocol.
  • the PrestoBlue Cell Viability Reagent (ThermoFisher, P50200) was used to assess cell viability of A549, H1792 and LCL-103H human cancer cell lines. 1000 cells were plated in 96-well plates in DMEM with 10% FBS. Next day the medium was changed for new media (DMEM with 0.5 % of FBS) and cells were incubated with inhibitors for 72 hours. The medium was then removed and PrestoBlue reagent was added to live cells, incubated for 45 minutes, then the fluorescence signal (ex:560 nm, em: 600 nm) was measured in a plate reader. Linearity of the detection was checked in a parallel experiment where control cells were serially diluted, and the signal was measured according to the manufacturer's protocol.
  • the human kinase specificity profile of BD837-IN-8 was tested by Reaction Biology Europe GmbH (Freiburg, Germany) using the Wild Type Kinase Panel comprised of 340 active human kinases. Briefly, profiling was done using the ’ ’ PanQi nascTM assay which measures the inhibition of phosphorylation of kinase substrate peptides in a radioactive assay format (Wang and Ma, 2015). The data is given as per cent remaining activity compared to the control experiment without the inhibitor. The inhibitor was used in 1 pM concentration and ATP was used at a concentration matching its apparent K M for each kinase.
  • Test compound, kinase, ATP, and a natural substrate peptide for each kinase were incubated in FlashPlate microtiter plates (PerkinElmer) coated with a scintillant. Reactions were stopped after one hour. Assays were carried out in duplicates.
  • HeLa cells were seeded into 24-well plate in DMEM supplemented with 10 % FBS. The next day the media was changed to DMEM.
  • PROTACs and/or the proteosome inhibitor (MG132) were added in 10 pM concentrations and cells were incubated for 24 hours. Cells then were lyzed in 100 p.L 1.5 X SDS loading buffer and samples (10 pL) were subjected to western blot analysis.
  • IRDye 680RD Goat anti -mouse IgG; Li-cor #926-68070; 1 : 10000
  • IRDye 800CW Goat anti-rabbit IgG, Li-Cor #926-32211; 1:5000.
  • the primary antibodies were the following: anti- JNK (Cell Signaling #3708; 1:3000), anti-p38 (Cell Signaling #9212; 1:3000 and anti-a-tubulin (Sigma #T6199;
  • Anti-a-tubulin antibody was used as the load control.
  • kinase inhibitors bind in the deep nucleotide binding crevice of protein kinases and block kinase activity.
  • These ATP-competitive drugs need to bind with high affinity (low nanomolar) as they compete against the millimolar concentration of ATP in the cell.
  • the presence of a surface cysteine on the target around the ATP -pocket may be exploited to increase binding affinity and target residence time, and thus inhibitor potency, by linking the noncovalent ATP-pocket binding moiety to a covalent warhead (Zhao et al., 2017) (Chaikuad et al., 2018).
  • the specificity of the composite inhibitor comes mostly from noncovalent contacts which are formed via the ATP-pocket binding moiety.
  • Protein kinases provide an excellent test system to compare currently used and new warhead scaffolds as cysteine attacking anchors for several reasons: 1) there are several well-documented examples of acrylamide warhead containing ATP-competitive inhibitors targeting these proteins (Boike at al, 2022), 2) the great structural similarity of members from this big family of enzymes allows comparative studies on specificity mechanisms, where negative selection against off-targets may be just as important as positive selection for the target, and 3) many kinases depend on intact protein-protein interactions mediated through shallow surface (docking) grooves that are difficult to target by small molecules (Miller et al, 2018). Protein kinases could be used to assess the benefits of new chemical warheads, which in turn could be applied on less-characterized protein targets.
  • the collection contained 75 compounds representing 7 types of Michael acceptors.
  • Each group contained ⁇ 5 to 20 different molecules where substituent groups varied: 2) cyclohexenone, 3) frustrated cyclohexenone, 4) acryl - chaicone, 5) cyanoacryl, 6) frustrated cyanoacryl and 7) nitroalkene.
  • the reactivity of the beta carbon in the different groups varies, as the chemical environment of a,P- unsaturated carbonyls affect the electronic properties of the warhead, while in frustrated compounds (Group 3 and 6) reactivity is also affected by steric hindrance.
  • the collection was used to search for molecules capable of interfering with the binding of fluorescently labeled D-motif peptides to ERK2, p38a or JNK1.
  • Group 3 (frustrated cyclohexenone) and Group 7 (nitroalkene) compounds showed interference with reporter D-motif peptide binding.
  • Group 7 compounds were found ineffective when the same screen was repeated under high amounts of GSH (10 mM), while, unexpectedly, Group 3 molecules retained their inhibitory effect.
  • Frustrated cyclohexenone compounds referred to as RU compounds with a specific number designation henceforth (e.g., RU 1 or RU46), were further tested.
  • Compounds contain an asymmetric center and we also measured MAPK binding of the different enantiomers in addition to the racemic mixtures used in former tests. Compounds were produced by stereoselective synthesis. Different stereoisomers with one chiral center bound to MAPKs with modest binding affinity difference ( ⁇ 5-fold), however, the stereoisomers of a molecule (RU70) containing two chiral centers (RU 73 -S'., S' and RU77-R,R) bound with ⁇ 8-10-fold difference (TABLE 2).
  • ester compounds e.g., RU58 - RU43, RU66 - RU72
  • other differently substituted amide compounds RU59, RU65, RU84, or RU85
  • Amide or oxazole containing compounds also formed a reversible covalent bond with ERK2 similarly to ester-containing warheads (FIGURE 4C).
  • TABLE 4 shows the details of protein crystallization, crystallographic data collection, and structure solution for the ERK2-RU67 covalent complex.
  • Bond angles (°) _ 0.859 _ a Crystals were grown with the vapor diffusion method and were frozen in liquid nitrogen in mother liquor supplemented with 25 % glycerol.
  • b Values in parenthesis are for the highest resolution bin. (hkl)- ⁇ I(hkl)>
  • such a multi-functionalized cyclohexenone scaffold has the right amount of reactivity towards the MAPK docking groove cysteine for reversible covalent bond formation but a hydrophobic substituent group (e.g., tert-butyl or benzyl) was also needed to direct the electrophile into the hydrophobic pocket next to the docking groove cysteine.
  • a hydrophobic substituent group e.g., tert-butyl or benzyl
  • the new cyclic warheads could be used in composite inhibitors in which the substituent groups may be extended to make additional noncovalent contacts to endow these more complex molecules (comprised of one of the new cyclic warhead scaffold designs and a directing group) with new properties, for example to increase binding towards selected cysteines and/or to achieve desirable cellular effects.
  • This positive selection mechanism is possibly the reason for the unexpectedly high efficiency of cyclohexenone-based warheads in targeting the MAPK D-groove cysteine.
  • the more complex ring structure can also hinder reactivity towards off-target cysteines due to higher probability for incompatibility with local surface topography (negative selection).
  • the nucleotide binding pocket of MAPKs is located close to the hydrophobic pockets of the D-groove (FIGURE 7A). It was shown earlier that connecting a D -peptide with an ATP pocket binding moiety can give a high-affinity bivalent ERK inhibitor (Lechtenberg et al., 2017).
  • the enantiomer of RU83 (referred to as RU 188) was linked to the ATP- binding moiety of an ATP competitive inhibitor, JNK-1N-8 (Zhang et al., 2012).
  • NanoBiT luciferase complementation protein-protein interaction assay concept was modified so that kinase docking facilitated phosphorylation of an artificial substrate sensor (D-sensor for MAPKs) could be qualitatively monitored in a microplate reader (Dixon et al., 2016).
  • This Phosphorylation-Assisted Luciferase Complementation assay (PhALC) was developed as a semi high-throughput, microplate compatible biochemical assay to obtain IC50 values of newly developed compounds on docking-based substrate phosphorylation.
  • the assay allows fast and cost-effective identification/characterization of any compound blocking MAPK activity (Poti et al, 2023).
  • RU83-click-pepMNKl_C contains the RU83 warhead extended towards the CD-groove. Based on the crystallographic models, the configuration of the substituent groups at C4 in this small molecule is better suited for extending the propargyl group towards the CD-groove (FIGURE 8A).
  • This compound was linked with the CD-groove binding region of a natural docking peptide (pepMNKl) and this RU83 -peptide chimera indeed efficiently blocked D- groove dependent phosphorylation in the presence of 10 mM GSH (where the lowest IC50 value, 0.4 pM, was found for p38oc, probably because this MAPK has the highest degree of structural plasticity at its docking groove).
  • MAPKs may also be blocked by inhibitors that target the ATP -binding pocket.
  • the configuration at C4 in RU188 is better suited for positioning IN-8 towards the ATP- pocket.
  • the IC50 for JNK1 was sub-micromolar (0.7 pM) even in the presence of 10 mM GSH (FIGURE 8B)
  • ester group at C4 could be extended by using azide-alkyne cycloaddition or Sonogashira coupling to add new groups directly to the cysteine targeting warhead moiety.
  • this group was modified with propargyl alcohol (RU83 and RU188), which were then fiirther extended by cycloaddition (RU103/RU189, RU115) or by Sonogashira coupling (RU140/RU141, RU214/215) (TABLE 5).
  • RU83 and RU188 propargyl alcohol
  • RU103/RU189, RU115 fiirther extended by cycloaddition
  • RU140/RU141, RU214/215 Sonogashira coupling
  • the binding affinity is also affected by the substituent group at C5.
  • RU78 which is a 5 -methyl derivative of RU60, bound weaker to ERK2 (and to the other MAPKs, too; see Table 2) because this somewhat bigger cyclohexenone derivative would likely be less compatible with the binding pocket topography around the cysteine-warhead adduct.
  • Structural comparisons between apoERK2, ERK2-D-peptide, and ERK2 -cyclic warhead complexes show that in the latter case, the hydrophobic pocket area of the D-groove widens to accommodate the cyclohexenone ring at (
  • RU60 forms a cysteine-adduct
  • RU187 forms a mixture (-50-50%) of cysteine(161)- and histidine(125)-adducts in the crystal structure.
  • This unexpected behavior of RU187 could only be explained by its different configuration which is likely not optimal in the chiral environment around cysteine(161), but it is a better fit for the nearby histidine(125), which unexpectedly is also a suitable nucleophile for the Michael-acceptor of the cyclic warhead.
  • cyclohexenone-based compounds are generally bound to p38oc better (see Tables 1-3 and 5). This could be explained by the more flexible overall structure of p38oc (Kumar et al, 2018): the docking groove of this MAPK accommodates an optimal cysteine- warhead adduct conformation easier, and/or noncovalent contacts mediated by the directing moiety can contribute more to overall binding energy.
  • MKK6 is the activator kinase of p38oc and the two proteins interact in a D-groove dependent manner (Garai et al, 2012) (Zeke eta al., 2015). This experiment showed that RU77 (used in 10 pM concentration) can block this binary interaction in live HEK293T cells.
  • TABLE 6 shows the details of protein crystallization, crystallographic data collection, and structure solution.
  • the enantiomer of RU68 is RU67, shown in Table 4; RU60/RU187 and RU75/RU76 are two other enantiomeric pairs).
  • the sulfhydryl group of a cysteine from a protein domain is in a more specific chemical environment: surface topography and the chemical nature of the neighboring amino acid sidechains will affect the intrinsic capacity of the a,p-unsaturated Michael acceptor (C3) to form the covalent bond.
  • C3 a,p-unsaturated Michael acceptor
  • the thiol of Cysll6 in JNK1 is more open and thus is more accessible compared to the MAPK D-groove cysteine (see Figure 5A). Therefore, this latter is a better test case to analyze the intrinsic reactivity of the new cyclic warhead scaffold design, because steric factors would be less disturbing at an “open” thiol position.
  • Cysteine reactivity of covalent warheads can be adjusted by modifying the electronic properties of the a,p- unsaturated carbonyl atom.
  • Acrylamide-based warheads form an irreversible covalent bond with noncatalytic surface cysteines, while installing a nitrile group, for example in cyanoacrylamide based warheads, the intrinsic reactivity of the olefin towards thiols increases but the formation of the irreversible adducts is eliminated.
  • Cyanoacrylamide based warheads had indeed been successfully applied to increase the occupancy of inhibitors originally developed to bind at the ATP -pocket of kinases (Serafimova et al, 2012)(Bradshaw et al, 2015).
  • keto groups may also be useful for increasing intrinsic reactivity combined with steric crowding to facilitate the elimination of Michael-donor amino acid residues (which could be referred to as “frustration” via sterical crowding).
  • the steric shielding around the reactive center of the cycloalkenone moiety can block the formation of stable adducts with hindered amino acid residues endowing higher site selectivity for the ligands and mitigating “off- target” binding.
  • the cyclic cyclohexenone/pentenone scaffolds provide a new platform: the electronic, steric properties, or ring strain could all be fine-tuned.
  • JNK-IN-8 acrylamide - acyclic irreversible
  • RU155 cyanoacrylamide
  • BD837, BD838, RU154, RU153, and RU172 acrylamide - acyclic irreversible
  • BD83837, BD838, RU154, RU153, and RU172 cyclohexenone/pentenone - cyclic reversible
  • the crystal structure of the JNK1-BD837-IN-8, -BD837-IN-8, and -RU135-IN-8 complexes were determined to reveal the structures of the Cysll6 covalent adduct (FIGURE 12 and TABLE 7).
  • the kinetic binding properties of the inhibitors were measured by SPR and a 2 -step reversible kinetic model was established for kinase - inhibitor binding (FIGURE 13A).
  • the reversible binding scheme included the formation of the noncovalent kinase- inhibitor (KI) complex as well as the covalent adduct (KI COV ).
  • association and dissociation of KI are described by the ki and k2 binding rates, while the formation of KI COV from KI or the formation of KI from KI COV are described by k 3 and k 4 , respectively.
  • kl and k2 were first independently determined using the cysteine to serine mutated protein (JNK1 Cysll6Ser) (FIGURE 13B,C).
  • K, (k 2 /ki) / (1+ (k 3 /k 4 )) in molarity (M) (Mons et al, 2022).
  • This analysis can also be used to calculate the energy contribution of reversible covalent bond formation for each inhibitor (AAG) (TABLE 8).
  • This comparative analysis showed that the reactivity of the cyclic warhead can be tuned via its substituent groups, and these can be used to set the binding affinity and to dramatically increase residence time (i.e. decrease k O ff) (FIGURE 13D).

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  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

De nombreux produits naturels biologiquement actifs contiennent des fragments électrophiles accepteurs de Michael. Par exemple, la curcumine et la 4-hydroxyderricine contiennent une cétone acyclique α,β-insaturée qui alkylate les cystéines. D'autres composés à base de plantes antitumoraux ou anti-inflammatoires, tels que la withaférine A ou le zérumbone, contiennent des cétones cycliques α,β-insaturées et réagissent avec des résidus nucléophiles de protéines. Ces observations ont contribué à un décalage de paradigme dans la conception et le développement de médicaments au cours des deux dernières décennies : divers médicaments à charge covalente ont été développés et approuvés. Malgré l'importance apparente et le succès des charges covalentes dans la conception et les développements actuels de médicaments, les charges appliquées présentent une variance structurale plutôt limitée et une complexité qui limite automatiquement l'espace chimique pouvant être atteint. En outre, pour réduire au minimum les réactions secondaires possibles pendant la synthèse de médicaments, les charges appliquées sont des appendices ajoutés dans l'étage tardif de la voie synthétique, ainsi un échafaudage de charge qui peut être modifié de manière synthétique facilement à l'aide de la chimie orthogonale et utilisé en tant que charge covalente accordable est encore manquant. Un tel échafaudage structuralement plus complexe serait beaucoup plus similaire aux charges des produits naturels et est censé être plus sélectif dans le ciblage de nucléophiles que l'on retrouve sur les protéines. De plus, grâce à une surface de contact plus grande, il pourrait être plus approprié pour cibler des surfaces protéiques peu profondes impliquées dans des interactions protéine-protéine.
EP23841041.9A 2022-11-15 2023-11-15 Échafaudages de concepteur cyclique pour le ciblage covalent de protéines par addition de michael Pending EP4619376A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
HUP2200449A HU231689B1 (hu) 2022-11-15 2022-11-15 Hangolható, reverzibilis kovalens kötés kialakítására képes, specifikus, ciszteint támadó c-Jun N-terminális kináz (JNK) inhibitorok
HU2300245A HUP2300245A1 (hu) 2023-07-10 2023-07-10 Ciklikus, desinger molekulavázak fehérjék Michael-addíciós kovalens célzásához
PCT/HU2023/050079 WO2024105420A2 (fr) 2022-11-15 2023-11-15 Échafaudages de concepteur cyclique pour le ciblage covalent de protéines par addition de michael

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EP4619376A2 true EP4619376A2 (fr) 2025-09-24

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2618613A1 (fr) * 2005-08-09 2007-02-22 Metaproteomics, Llc Modulation des proteines kinases a l'aide de produits a base de houblon ou d'acacia
DE102005052501A1 (de) * 2005-11-03 2007-05-16 Univ Ernst Moritz Arndt Neue Leitstrukturen für zytostatische Verbindungen auf Basis von Spiroverbindungen
US9382239B2 (en) 2011-11-17 2016-07-05 Dana-Farber Cancer Institute, Inc. Inhibitors of c-Jun-N-terminal kinase (JNK)
JP7250738B2 (ja) 2020-07-22 2023-04-03 信越化学工業株式会社 ３，３－ジメチル－１－ブテン－１，４－ジカルボキシレート化合物及び１，３，３－トリメチル－１－ブテン－１，４－ジカルボキシレート化合物、並びに、これらを用いた５，５－ジメチル－２－オキソ－３－シクロペンテン－１－カルボキシレート化合物及び３，５，５－トリメチル－２－オキソ－３－シクロペンテン－１－カルボキシレート化合物の製造方法

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