WO2014170403A1 - Kinase inhibitors - Google Patents

Abstract

There is provided inter alia a compound of formula (I), Q-L-P wherein Q is a moiety that binds to the ATP -binding site of a Pim kinase; L is an organic tether that connects Q to P and permits simultaneous binding of Q and P to a Pim kinase; P is a moiety that binds to the protein binding domain of Pim kinase; and wherein the compound of formula (I) is optionally labelled with a fluorophore.

Description

KINASE INHIBITORS

Field of the Invention

This invention relates to protein kinase inhibitors and methods of preparing them and their use. In particular, the invention relates to inhibitors of Pim kinases that simultaneously bind to the ATP -binding site and the protein substrate-binding domain of these kinases.

Background of the Invention

Protein kinases are enzymes that catalyze phosphorylation of proteins. They are key regulators of cellular protein phosphorylation balances. Aberrant activity of protein kinases is the cause of several serious deseases like cancers, diabetes and atherosclerosis. Over the past 15 years protein kinases have become the pharmaceutical industry's most important class of drug target in the field of cancer. More than 20 drugs that target kinases have been approved for clinical use over the past 12 years, and hundreds more are undergoing clinical trials. [Cohen P et al. ACS Chem Biol. 2013;8:96-104; Knapp S et al. Nat Chem Biol. 2013;9:3-6].

The Pim (Provirus Integration site for Moloney murine leukemia virus) family of protein kinases (PKs) includes 3 constitutively active serine/threonine kinases. At the protein amino acid level Pim-2 and Pim-3 show 61 and 71% identity to Pim-1, respectively [Mikkers H et al. Mol Cell Biol. 2004;24: 104-115]. Pim kinases participate in several normal biological processes including cell survival, proliferation, differentiation and apoptosis. Elevated expression levels of Pim-1 and Pim-2 have been observed in hematologic malignancies and prostate cancer, increased Pim-3 expression has been also detected in solid tumours [Blanco-Aparicio C et al. Biochem

Pharmacol. 2013;85:629-643]. These findings point to the Pim kinases as proto-oncogenes that are important for the initiation and progression of human cancer. The potentiality to use regulators of Pim kinase activity for fighting with cancer brought a small-molecule inhibitor SGI- 1776 to the first stage of clinical trials with refractory prostate cancer and

relapsed/refractory non-Hodgkin's lymphoma. SGI- 1776 possesses inhibition IC₅₀ values of 7 nM, 363 nM, 69 nM toward protein kinases Pim-1, Pim-2 and Pim-3, respectively. It also possesses comparative inhibitory potency (IC₅₀ = 44 nM) towards protein kinase Flt-3. This off- target effect may be one of the reasons of failure of SGI- 1776 in first stage clinical trials (SGI- 1776 was withdrawn from clinical development because of identification of the dose-limiting cardiotoxicity of the compound) [Blanco-Aparicio C et al. Biochem Pharmacol. 2013;85:629- 643].

The pharmaceutical company AstraZeneca is developing the pan-Pirn kinase inhibitor AZD1208 for the potential treatment of cancer. In March 2012, a phase I trial in AML patients was initiated to assess the safety, efficacy and pharmacokinetics of this drug [Blanco-Aparicio C et al.

Biochem Pharmacol. 2013;85:629-643].

Pim kinases are regulated by Jak/Stat pathway that leads to activation of Pim genes and synthesis of the proteins. Pim kinases are constitutively active and no further post translation modification is needed for Pim kinase activity. Thus, signalling is largely regulated on both the transcriptional/translational level and the protein turnover level [Blanco-Aparicio C et al. Biochem Pharmacol. 2013;85:629-643].

Pim kinases realize their oncogenic activities through the regulation of MYC transcriptional activity, the regulation of cap-dependent protein translation, the regulation of cell cycle progression, through survival signalling by phosphorylation of BAD, and other biochemical mechanisms [Nawijn MC et al. Nat Rev Cancer. 2011;11 :23-34].

Inhibition of the Pim kinases leads to inhibition of proliferation and survival of multiple cancer cell types. Therefore this would be expected to provide a therapeutic benefit to cancer patients with a variety of cancers as well as other conditions that are mediated by Pim kinase signalling pathway.

Aberrant Pim kinase activity has been related also to non-cancer diseases. PIM kinases have recently been identified as regulators of human primary T helper cell differentiation, thus pointing to a new possibility for development of therapeutics for many autoimmune and allergic diseases. [Tahvanainen J et al. J Biol Chem. 2013;288:3048-3058]

In hematopoietic malignancies and in a variety of solid tumors, increased Pim- 1 expression has been shown to correlate with the stage of disease. This characteristic suggests that expression level and activity of Pim kinases can serve as a useful biomarkers for cancer diagnosis and prognosis [Magnuson NS et al. Future Oncol. 2010:6: 1461 - 1478].

Structurally different small-molecule inhibitors (indolocarbazoles, bisindolyimaleimides, naphthyridines, pyridazines and isoxazoles to thiazolidine-2,4-diones, thienopyrimidinones, pyridones and isoxazoloquino lines [Merkel AL et al. Expert Opin Investig Drugs. 2012;21 :425- 36) have been developed for regulation of activity of Pim kinases both as research tools and drug candidates. More than 400 small molecular Pim- 1 inhibitors with A^', (IC₅₀) values < 10 μΜ have been registered in the ChEMBL database [Ogava N et al. Expert Opin Drug Discov.

20127: 1177-1192]. The developments in this field have been described in numerous scientific papers and patent applications (Morwick T. Expert Opin Ther Pat. 2010;20: 193-212; Blanco- Aparicio C et al. Biochem Pharmacol. 2013;85:629-643).

The great majority of reported Pim inhibitors target the ATP-binding site of the enzymes. The introduction of compounds possessing their inhibitory properties via binding to the binding site of ATP into medical practice as drugs has great risks because the high selectivity of the interaction is difficultly achievable and controllable. As all human 518 protein kinases use ATP as a co-substrate [Manning G et al. Science 2002;298: 1912-1934], many previously reported selective inhibitors of Pim kinases have later turned out to realize their physiological effect through inhibition of other enzymes. The inhibitory potency of great majority of disclosed Pirn inhibitors has been characterized towards a very limited number of protein kinases. Therefore results that are unexpected and difficult to interpret are very common in Pirn research.

In addition to protein kinases, more than 2500 other proteins, coded by 13% of human genes (protein kinases, small G proteins, dehydrogenases, ATPases, helicases, non-convent ional purine-utilizing proteins, synthetases, purinergic receptors, etc.), belong to the human purinome. Ail these proteins (all together 3266 proteins) possess a purine-binding site and thus are potential off-targets of inhibitors binding to the ATP -binding site of a protein kinase. [Knapp S et al., Curr. Top. Med. Chem. 2006.6: 1 129 ]. Thus the work-out of selective inhibitors for biomedical research and drug development is connected to great risks because of impossibility of testing of compounds under development with all of more than 3000 purine-binding proteins.

One way to increase the selectivity and inhibitory potency of an inhibitor is to widen the area of contacts of the inhibitor with the target protein by increasing the number of interactions between the interacting partners by including amino acid residues from outside the conserved purine- binding pocket of the kinase to binding with the inhibitor. Factually, the protein substrate- binding domain of protein kinases is less conserved than the ATP -binding site, thus compounds whose binding to the kinase in addition to the interaction with the ATP-binding pocket includes interactions with the former domain (e.g., bisubstrate inhibitors) have great potential to be highly potent and group-selective inhibitors of protein kinases. The bisubstrate approach, i.e.

construction of inhibitors that simultaneously associate with binding areas of both substrates of a protein kinase - ATP and the substrate protein, has been used to develop highly potent and group-selective inhibitors of protein kinases [Lavogina D et al. ChemMedChem. 2010;5:23-34]. Thus conjugation of moderately affinity (KD values in micromolar range) ATP-competitive inhibitors with peptides containing 2-10 arginine residues (ARCs - conjugates of nucleoside analogues and peptides) has led to inhibitors that possess high affinity (K_D values in picomolar or low nanomolar range) towards many basophilic protein kinases, mostly belonging to the AGC group [Manning G et al. Science 2002;298:1912-1934] of protein kinases [Lavogina D et al. J Med Chem. 2009;52:308-321 ; Lavogina D et al. Bioorg Med Chem Lett. 2012;22:3425-3430, WO2008/019696, US8158376 B2, IN253208], thus leading to generic inhibitors of protein kinases.

Pirn kinases belong to the Ca2+/calmodulin-dependent protein kinase (CAMK) group [Manning G et al. Science 2002;298: 1912-1934]. The established consensus sequence of the Pirn kinases (K/R)3-X-S/T-X, with X being neither a basic nor a large hydrophobic residue, points to the basophilic character of the Pirn kinases [Blanco -Aparicio C, et al. Biochem Pharmacol.

2013;85:629-643]. In line with this quality of Pirn kinases, some inhibitors that inhibited other basophilic protein kinases (mostly belonging to the AGC group) also inhibited Pirn kinases (belonging to CAMC group) with comparable potency (as determined in a panel of protein kinases) [Enkvist E et al. J Med Chem. 2006;49:7150-7159; Lavogina D et al. J Med Chem. 2009;52:308-321]. None, however, of the previously characterized bisubstrate inhibitors

(published and unpublished data) pointed to structural elements in conjugates of nucleoside analogues and arginine-containing peptides that improved Pirn kinase-selectivity of the compounds.

Three methods can be used to prove the bisubstrate character of a protein kinase inhibitor

[Lavogina et al. ChemMedChem. 2010; 5:23-34]:

1. Analysis of the inhibitor-PK co-crystal structures;

2. Kinetic analysis of the competitiveness of the inhibitor versus either substrate; and

3. Displacement of the inhibitor from its complex with PK by both the ATP-competitive and protein substrate-competitive inhibitors.

Additional evidence pointing to the bisubstrate character of inhibitors may come from the selectivity patterns and increased inhibitory potency of conjugates pointing to the interaction of the inhibitor with binding sites of both substrates.

The bisubstrate approach has been previously used for the development of potent inhibitors of PKs (reviews: Parang et al. Pharmacol Ther. 2002 ,93: 145-157; Lavogina et al. ChemMedChem.

2010; 5:23-34). Conjugates of isoquinolinesulfonamides and peptides were successfully used for the inhibition of PKA and PKC (Ricouart et al. J. Med. Chem. 1991;34:73-78). Later, conjugates of adenosine analogs and peptides were used for inhibition of PKA and PKC (Loog et al. Bioorg.

Med. Chem. Lett. 1999;9: 1447-1452). The latter approach used by authors of the present invention has thereafter given inhibitors whose inhibitory potency goes into pico molar range for basophilic PKs [Lavogina D et al. Bioorg Med Chem Lett. 2012;22:3425-3430] of the AGC group [Manning G et al. Science. 2002;298:1912-34] .

Peptide- ATP analogs as PKA inhibitors have also been described, wherein ATP is linked to a protein kinase peptide substrate. WO 01/70770 discloses inhibitors for the insulin receptor tyrosine kinase comprising an ATPgamma-S linked to a peptide substrate analog via a two- carbon spacer. This approach has a limitation for the development of inhibitors usable in cellular studies because of the transport and stability properties of the conjugates. WO03010281 relates to specific isoquinoline conjugates with peptides which are PKB inhibitors. Recently the bisubstrate approach has been successfully applied by authors of the present invention for construction of potent and rather selective inhibitors for acidophilic protein kinase CK2

[European patent EP2700946; Enkvist E et al. Org Biomol Chem. 2012;10: 8645-8653]. Authors of the present invention have also successfully used the bisubstrate approach for development of affinity adsorbents for protein kinases [European patent EP1179050], fluorescent probes for protein kinases [US patent US8158376], pegylated inhibitors for protein kinases [Estonian patent EE05396 Bl], responsive microsecond-scale photo luminescent probes for protein kinases

[European patent EP2482072] and fluorescent probes for protein kinase CK2 [European patent EP2700946]. Since 2005 numerous crystal structures of ligand/Pim-1 complexes have pointed to the uniqueness of a network of hydrogen bonds and hydrophobic interactions important for binding of the co-substrate ATP (and its analogues) and ATP-competive inhibitors to Pirn kinases

[Blanco-Aparicio C et al. Biochem Pharmacol. 2013;85:629-643]. Most human 518 protein kinases form two hydrogen bonds with the adenine part of ATP through residues in the hinge region, while Pim-1 (and other Pirn kinases) forms only one hydrogen bond, between the N6 atom of adenine and the backbone carbonyl oxygen atom of the first hinge residue (Glul21). Other kinases additionally form a hydrogen bond between the Nl atom of adenine and the backbone NH of the third hinge residue. The proline residue at position 123 in Pim-1 precludes the kinase from this capability.

The previously established (Palaty CK et al. Biochem. Cell Biol. 1997;75: 153-162) Pim-1 consensus substrate peptide sequence K/R-K/R-R-K/R-L-S/T-a reveals a strong basic environment at the amino terminal side of Pirn substrate proteins and peptides. As a part of the present invention we showed that arginine-rich peptides could possess remarkably good affinity to the Pirn kinases (IQ of 290 nM was determined for oligoarginine peptide (D-Arg)c_>-NH₂ towards Pim-1). This affinity was more than 100-fold higher than that of a representative of basophilic kinases of the AGC group, catalytic subunit of cAMP-dependent protein kinase (PKAc), that revealed ¾ value of 35 μΜ for (D-Arg)₉-NH₂.

In the present invention this distinctness in the Pirn protein structure, together with the remarkably high affinity of Pim-1 kinase to arginine-rich peptides (compared to other basophilic protein kinases) established here was used for the construction of highly potent selective inhibitors for Pirn kinases.

Oligo-arginines comprising more than 6 arginine (L-arginine or D-arginine) residues are acknowledged transport peptides [Nakase I et al. Mol Biosyst. 2013;9:855-861] and

incorporation of such fragments into bioactive conjugates makes them cell plasma membrane permeable and enables the application these conjugates in living cells, tissues and organisms.

Brief summary of the Invention

The current invention provides compounds having high Pim-selectivity and high Pirn inhibitory potency whose development was based on the application of bisubstrate approach. Novel inhibitors were developed based on selection of fragments binding to the atypical ly structured adenosi ne-b i n d i ng pocket of Pirn kinases and conjugation of the fragments with peptides containing multiple arginine residues through a cyclic spacer and an organic linker. The optimizat ion of the structure of the linker enabled the development of compounds where the simultanous association of the inhibitor with both the ATP-binding pocket and the substrate protein-binding domain of the Pirn kinase was possible leading to high (pico molar) affinity and good Pirn kinase selectivity of the inhibitors.

As a first aspect of the invention there is provided a compound of formula (I): Q-L-P (I)

Q is a moiety that binds to the ATP -binding site of a Pim kinase;

L is an organic tether that connects Q to P and permits simultaneous binding of Q and P to

Pim kinase; and

P is a moiety that binds to the protein substrate binding domain of a Pim kinase;

wherein the compound of formula (I ) is optionally labelled with a fluorophore.

Definitions

Peptidic fragment refers to a moiety of a compound that is formed of a short chain (typically less than 50) of amino acid monomers (natural (including the 20 standard proteinogenic amino acids) or non natural, and including D and L isomeric derivatives thereof) linked by peptide (amide) bonds.

Peptidomimetic fragment refers to a moiety of a compound that contains non-peptidic structural elements and that is capable of inducing similar biochemical effects as the parent peptide.

Fluorophore means a fluorescent chemical that can re-emit light upon light excitation.

Brief description of the Figures

Figure 1 shows the result of testing the displacement of ARC- 1139 from its complex with Pim-1 by various test compounds, as determined by measurement of luminescence intensity (see Example 46)

Figure 2 shows the results of Omnia kinetic assay testing for compounds against Pim-1 kinase (see Example 47)

Figure 3 shows the titration of ARC-3158 with Pim-1 as measured by luminescence intensity (see Example 48)

Detailed description of the Invention

In an embodiment, the Pim kinase to which compound of formula (I) binds is selected from Pim- 1 kinase Pim-2 kinase and Pim-3 kinase, particularly Pim-1 kinase.

In an embodiment, Q comprises or consists of an aromatic or hetero aromatic ring system comprising two fused rings (bicycle), for example a 5-6 fused cycle, or 3 fused rings (tricycle), for example a 6-5-6 fused ring system. In an embodiment, Q comprises or consists of thiophene, selenophene or tellurophene fused to one or two 6 membered rings (e.g. as the central ring of a 6-5- tricycle), at least one of said one two 6 membered rings suitably containing one or two N atoms. In an embodiment Q represents an analogue of a thiophene, selenophene or tellurophene ring, said analogue containing one or two N atoms in place of ring carbon atoms, fused to one or two 6 membered rings, at least one of said one two 6 membered rings suitably containing two N atoms.

In particular, Q may represent a moiety of formula (Al):

wherein

Ri is Br, CI, I, or H;

R₂ is Br, CI, I, H or OH;

R₃ is CH or N;

K represents a link such as -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

p represents 0 or 1, typically 0;

q represents 0 or an integer 1-6, typically 0 or 1;

r represents 0 or an integer 1-6, typically 0 or 1;

R₄ represents the point of attachment to L-P;

R₅ is O, S, Se, Te, NH, N-CH₃.

In an embodiment of formula (Al), p represents 0.

Altern

wherein

Ri is Br, CI, I, or H;

R₂ is Br, CI, I, H or OH;

R₃ is CH or N;

K represents a link such as -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

p represents 0 or 1, typically 0;

q represents an integer 1-6, typically 1;

r represents 0 or an integer 1-6, typically 0 or 1;

R₄ represents the point of attachment to L-P;

R₅ represents O, S, Se, Te, NH or N-CH₃;

Je represents a 5-7 membered heterocyclic ring containing 1 or 2 heteroatoms selected from O, N and S;

z represents 0 or 1 , typically 1 ; said moiety of formula (A2) being optionally substituted on one or more of the aromatic rings by one or more (e.g. one) halogen groups, particularly selected from Br and I.

In an embodiment, Je represents a pyrrolidine or piperidine ring, suitably connected to the rest of the molecule via its N atom.

In an embodiment of formula (A2), p represents 0.

In a specific embodiment of moiety (A2), Q may represent a structure of (Α2')

wherein R_{l s} R₂, R₃, R₄, R5, K, p and Je are as defined for structure (A2).

In an embodiment of moieties of formula (Al), (A2) or (Α2'), Rs represents S.

In another embodiment of moiety (Al), (A2) or (Α2'), Rs represents Se.

In an embodiment of moieties of formula (Al), (A2) or (Α2'), Ri represents H.

In an embodiment of moieties of formula (Al), (A2) or (Α2'), R₂ represents Br.

In an embodiment of moieties of formula (Al), (A2) or (Α2'), R₃ represents CH.

Alternatively, Q may represent a moiety of formula (Bl) or (B2):

wherein X represents Se or Te, suitably Se;

K represents a link such as -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

p represents 0 or 1, typically 0;

q represents an integer 1-6;

r represents 0 or an integer 1-6, typically 0 or 1;

R₄ represents the point of attachment to L-P;

said moiety of formula (Bl) or (B2) being optionally substituted on one or more of the aromatic rings by one or more (e.g. one) halogen groups, particularly selected from Br and I.

Alternatively Q represents a moiety of formula (C):

(C) wherein

Ri is Br, CI, I, or H;

R₂ is Br, CI, I, H, or OH;

R₃ is CH or N;

R₄ represents the point of attachment to L-P;

R₅ is O, S, Se, Te, NH, N-CH₃.

In an embodiment of a moiety of formula (C), R₅ represents S. In an alternative embodiment of a moiety of formula (C), R₅ represents Se.

In an embodiment of a moiety of formula (C), Ri represents H.

In an embodiment of a moiety of formula (C), R₂ represents Br.

In an embodiment of a moiety of formula (C), R₃ represents CH.

Alternatively, Q represents a moiety of formula (D):

wherein K represents a link such as -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

p represents 0 or 1, typically 0;

q represents an integer 1-6;

r represents 0 or an integer 1-11 e.g. 1-6, typically 0 or 1;

R₄ represents the point of attachment to L-P;

said moiety of formula (D) being optionally substituted on the phenyl ring by one or more (e.g. one) halogen groups, particularly selected from Br and I.

In an embodiment, in a moiety of formula (D) the phenyl ring is substituted by four halogen atoms, especially by four I atoms.

Alternatively, Q represents a moiety of formula (E):

(E)

wherein K represents a link such as -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

p represents 0 or 1 , typically 1 ;

Z represents CH or N; q represents 0 or an integer 1-6, typically 0, save when Z is N in which case it has a minimum value of 1 ;

r represents 0 or an integer 1-6, typically 1;

R4 represents the point of attachment to L-P;

said moiety of formula (E) being optionally substituted on one or both aromatic rings by one or more groups (e.g. one group) selected from halogen, Cl-4alkyl (e.g. methyl) or -CHO.

Alternatively, Q represents a moiety of formula (F):

wherein K represents a link such as -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

p represents 0 or 1, typically 0;

q represents an integer 1-6;

r represents 0 or an integer 1-11, e.g. 1 to 9 such as 1 to 5, typically 0 or 1;

R4 represents the point of attachment to L-P;

said moiety of formula (F) being optionally substituted on the phenyl ring by one or more (e.g. one) halogen groups, particularly selected from Br and I.

In an embodiment said moiety of formula (F) is substituted on the phenyl ring by halogen atoms, especially by four I atoms. Alternatively, Q represents a moiety of formula (G):

wherein X represents Se or Te, suitably Se;

K represents a link such as -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

p represents 0 or 1, typically 0;

q represents an integer 1 to 6;

r represents 0 or an integer 1-6, typically 0 or 1;

P4 represents the point of attachment to L-P;

Ph represents phenyl which may optionally be substituted by one or more (e.g. 1 or 2) substituents selected from Cl-4alkyl (e.g. methyl) and halogen; said moiety of formula (G) being optionally substituted on one or more of the aromatic rings by one or more (e.g. one) halogen groups, particularly selected from Br and I.

Alternatively, Q represents a moiety of formula (H):

wherein X represents Se or Te, suitably Se;

W represents CH₂ or NH, suitably NH;

K represents a link such as -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

p represents 0 or 1, typically 0;

q represents 0 or an integer 1-6, typically 0 or 1,

r represents 0 or an integer 1-6, typically 0 or 1;

R4 represents the point of attachment to L-P;

said moiety of formula (H) being optionally substituted on one or more of the aromatic rings by one or more (e.g. one) halogen groups, particularly selected from Br and I.

Alternatively, Q represents a moiety of formula (J):

wherein X represents Se or Te, suitably Se;

K represents a link such as -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

p represents 0 or 1 , suitably 1 ;

q represents an integer 1-6;

r represents 0 or an integer 1-6, suitably 0;

said moiety of formula (J) being optionally substituted on one or more of the aromatic rings by one or more halogen groups;

In structures A2, Bl, B2, D, E, F, G, H and J, K (if present) represents a link such as -(CH₂)_q- OC(O)- or -(CH₂)_r-C(0)-; more generally the link may, for example, be an aliphatic carbon chain e.g. a Cl-10 carbon chain (straight or branched, preferably straight chain) (e.g. a CI -8 or Cl-6 or Cl-4 carbon chain) in which one or more carbon atoms (e.g. 1-4 e.g. 1 or 2) carbon atoms are optionally replaced with heteroatoms selected from O, N, S, SO or S0₂ (e.g. O or S) and in which one or more carbon atoms are optionally replaced with C(O). In an embodiment, P represents a peptidic fragment incorporating one or more arginine residues or a peptidomimetic fragment incorporating one or more sidechain guanidine groups.

In an embodiment, P includes two or more D-amino acid residues.

In an embodiment, P includes a (D-Arg)_n moiety wherein n is 2-10.

In an embodiment, P includes two or more guanidine groups.

In an embodiment, P includes a terminal Lys residue e.g. a terminal D-Lys residue.

In an embodiment, myristic acid or another hydrophobic fatty acid may be attached to a free amino group in the P m iety, for example attached to the sidechain amine of a lysine residue such as a terminal lysine moiety. Such derivatives may have enhanced cell membrane penetration ability. Thus, in an embodiment, myristic acid is attached via its acid group to the sidechain amine of a lysine residue in the P moiety.

In an embodiment, P includes a (D-Arg)₆-(D-Lys)- moiety, for example a terminal (D-Arg)₆-(D- Lys)-NH₂ moiety. The length of L will be suitable to permit simultaneous binding of Q and P into their respective binding pockets in the Pirn kinase (i.e. the ATP binding site and the protein substrate binding domain). Suitably L is connected to Q and to P via amide bonds. In an embodiment, L includes the residue of the amino acid H₂N-(CH₂)_m-COOH in which m represents an integer 3 to 10, preferably 6-8, such as aminohexanoic acid or aminooctanoic acid. Thus, in an embodiment, L includes an Ahx moiety.

In an embodiment, L includes the residue of a cyclic amino acids (including amino acids of natural or unnatural origin), such as a Hyp residue, especially trans-Hyp.

In an embodiment, L includes a motif selected from Hyp, Hyp-Ahx, Hyp-Aox and Ahx-(D-Arg)- Ahx and suitably each Hyp is trans-Hyp.

Compounds of formula (I), particularly moieties L and P and especially P, may contain amino acid residues in D- or L-configuration. Suitably at least two D amino acids are included.

Suitable all amino acids are D amino acids. Use of D-amino acids tends to increase the proteolytic stability of the compound of formula (I) as compared with use of L-amino acids and is thus desirable.

In an embodiment, L-P represents a moiety selected from:

Ahx-(D-Arg)₆-(D-Lys)-NH₂;

Ahx-(D-Arg)-Ahx-(D-Arg)₆-(D-Lys)-NH₂;

Hyp -Ahx-(D-Arg)₆-(D-Lys)-NH₂;

Hyp-Ahx-(D-Arg)₂-NH₂;

Hyp-Aox-(D-Arg)₂-NH₂;

Hyp-Ahx-(D-Arg)-Ahx-(D-Arg)₆-(D-Lys)-NH₂;

Hyp-Ahx-(D-Arg)₄-NH₂;

Hyp-Ahx-(D-Arg)₈-NH₂; Hyp-(D-Arg)8-NH₂;Suitably compounds of formula (I) show good cell penetration ability.

Suitably compounds of formula (I) obey Lipinski's rule of 5.

Pharmaceutical uses

The invention provides a compound of formula (I ) for use as a pharmaceutical, especially for use in the treatment or prevention of a condition associated with activity of a Pirn kinase, such as cancer.

Relevant cancers include hematologic malignancies (including non-Hodgkin's lymphoma) and solid tumours such as prostate cancer. Narlik-Grassow M et al. Cancer. Med Res Rev. 2013 Apr 10. doi: 10.1002/med.21284 lists human cancers where the increased expression of any of the three Pirn kinases has been determined (in brackets, over-expressed genes): B-cell chronic lymphocytic leukemia (PIM1;PIM2); Mantle cell lymphoma (PIM1;PIM2); DLBCL (PIM1; PIM2); Butkitt's lymphoma (PIM1); B-cell lymphoma (PIM1; PIM2); Acute myeloid leukemia (PIM2); Prostate cancer (PIM1; PIM2); Bladder carcinoma (PIM1); Pancreatic cancer (PIM1; PIM3); Gastric carcinoma (PIM1; PIM3); Squamous cell carcinoma of head and neck (PIM1); Oral squamous carcinoma (PIM1); Colorectal carcinoma (PIM1;PIM3); Liver cancer (PIM1); Liposarcoma (PIM1). Accordingly compounds of formula (I) may be useful to treat or prevent the aforementioned conditions.

Although cancer appears to be the primary focus of Pirn kinase inhibitor-based drugs, many other diseases, like inflammatory diseases, immune suppression, degenerative diseases, ischemia, reperfusion injury, autoimmune diseases, allergic reactions, organ transplantation rejection, cardiovascular diseases, rheumatoid arthritis, diabetic retinopathy, neurodegenerative diseases, diabetes, autoimmune diseases, viral infection have been disclosed as potential targets of such drugs [Morwick T. Expert Opin Ther Pat. 2010;20: 193-212]. Accordingly compounds of formula (I) may also be useful to treat or prevent the aforementioned conditions.

The invention provides a method of treatment or prevention of a condition associated with activity of a Pirn kinase, such as cancer, which comprises administering to a patient in need thereof a pharmaceutically effective amount of a compound of formula (I).

The invention provides a use of compound of formula ( I ) in the manufacture of a medicament for the treatment or prevention of a condition associated with activity of a Pirn kinase, such as cancer.

The invention provides a pharmaceutical composition comprising a compound of formula (I) and one or more pharmaceutically acceptable diluents or carriers.

Diluents and carriers may include those suitable for parenteral, oral, topical, mucosal and rectal administration.

Such compositions may be prepared e.g. for parenteral, subcutaneous, intramuscular,

intravenous, intra-articular or peri-articular administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; for topical e.g. intravitreal, pulmonary or intranasal administration, particularly in the form of eye drops, powders, nasal drops or aerosols and transdermal administration; for mucosal administration e.g. to buccal, sublingual or vaginal mucosa, and for rectal administration e.g. in the form of a suppository.

The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA., (1985). Formulations for parenteral administration may contain as excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Formulations for nasal administration may be solid and may contain excipients, for example, lactose or dextran, or may be aqueous or oily solutions for use in the form of nasal drops or metered spray. For buccal administration typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinated starch, and the like.

Compositions suitable for oral administration may comprise one or more physiologically compatible carriers and/or excipients and may be in solid or liquid form. Tablets and capsules may be prepared with binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or poly-vinylpyrollidone; fillers, such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine; lubricants, such as magnesium stearate, talc, polyethylene glycol, or silica; and surfactants, such as sodium lauryl sulfate. Liquid compositions may contain conventional additives such as suspending agents, for example sorbitol syrup, methyl cellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or edible fats; emulsifying agents such as lecithin, or acacia; vegetable oils such as almond oil, coconut oil, cod liver oil, or peanut oil; preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Liquid compositions may be encapsulated in, for example, gelatin to provide a unit dosage form.

Solid oral dosage forms include tablets, two-piece hard shell capsules and soft elastic gelatin (SEG) capsules.

A dry shell formulation typically comprises of about 40% to 60% concentration of gelatin, about a 20%) to 30%) concentration of plasticizer (such as glycerin, sorbitol or propylene glycol) and about a 30%> to 40%> concentration of water. Other materials such as preservatives, dyes, opacifiers and flavours also may be present. The liquid fill material comprises a solid drug that has been dissolved, solubilized or dispersed (with suspending agents such as beeswax, hydrogenated castor oil or polyethylene glycol 4000) or a liquid drug in vehicles or combinations of vehicles such as mineral oil, vegetable oils, triglycerides, glycols, polyols and surface-active agents.

Pharmaceutical compositions of the invention may optionally include one or more anti-oxidants (e.g. ascorbic acid or metabisulfate and salts thereof). Fluorophore containing compounds and further uses

The compound of the invention may optionally be label led with a fluorophore, the fluorophore being incorporated into Q, L or P, more suitably into L or P and especial ly P. Such compounds may be represented as having formula (Q-L-P )-(·^'. Moiety F may optional ly incorporate a linker to facilitate linkage of the fluoraphorc to the rest of the molecule. Such compounds retain their activity as Pim. kinase inhibitors. Example fluorophores F include fluorescein, FITC, rhodamine, 6-FAM, TET, HEX, Cy3, Cy3B, TMR, ROX, Texas Red, Cy5, Cy7, Alexa Fluor dyes (e.g., Alexa 647), BODIPY dyes, PromoFluor dyes (e.g., PromoFluor 555 and PromoFluor 647), Atto- Tec dyes (e.g., ATTO 647N and ATTO 740), DyLight dyes, SureLight dyes, IRDye 700, IRDye 800.

Fluorophores may, for example, be incorporated via coupling to a D-Lys residue (for example a terminal D-Lys residue) in P.

Thus the invention provides use of a compound of formula (I) in an assay for determining the activity or amount of a Pim kinase inhibitor.

The invention provides use of a compound of formula (I) as a fluorescent or photoluminescent probe.

The invention al o prov ides a compound of formula (I) incorporating a fluorophore may be used i an assay for the ident ificat ion of a Pim kinase inhibitor by its displacement of a compound of formula (I) from its complex with the Pim kinase by an inhibitor compound present in the solution comprising the steps of:

1) optionally establishing the K_ti for the bound complex of the compound of formula (I) incorporating a fluorophore with the Pim kinase;

2) contacting the compound of formula (I) incorporat ing a f uorophore with the Pim kinase and measuring the fluorescence signal of the formed complex;

3) incubat ion of the complex formed in the previous step with the potential inhibitory compound or a mixture of compounds and measuring of the fluorescence signal of the compound of formula (I) incorporating a fluorophore in its complex with the Pim kinase;

4) comparing the fluorescence signals from step 2) and step 3), a reduction in the fluorescence signal of step 3 ) as compared w ith that of step 2) indicating that the potential inhibitory compound or a mixture of compounds is capable of binding to the Pim kinase.

The fluorescent label led compounds of formula (I) („probe") can be used in biochemical studies for determination of Pim kinase concentration (especially in act ive form) and characterization of compet it ive inhibitors in bind i ng d i sp I acement assays. The probe can be also used for mapping of Pim kinases in living cells. Additionally, in hematopoiet ic mal ignancies and in a variety of solid tumours, increased Pim- 1 expression has been shown to correlate with the stage of disease. This characterist ic suggests it can serve as a useful biomarker for cancer diagnosis and prognosis [Magnuson NS et al. Future Oncol. 2010;6: 1461-1478]. Therefore high affinity fluorescent probes (inhibitors labeled with ffuorophores) could be useful tool for the measurement of Pirn protein expression level in tissues and cells (e.g., blood cells).

Recently, authors of the present patent discovered a new phenomenon, protein- induced long lifetime luminescence of non-metal probes [European application EP1 1 152289; Enkvist E et al. ACS Chem Biol 201 1 ;6: 1052-1062]. ARC-Lum probes incorporate a thiophene, a selenophene or a tellurophene heterocycle and a fluorophore conjugated to the lysine residue in the peptide fragment. In the complex with a PK, ARC-Lum probes emit long-lifetime (microsecond-scale) luminescence at the emission wavelengths of the fluorescent label if the complex is illuminated at the excitation wavelength of the thiophene-, selenophene- or tellurophene-containing phosphorescence donors. Some the compounds of the present invention also have optical properties of ARC-Lum probes. They possess long lifetime emission signal when they are bound to Pirn kinases.

Responsive ARC-Lum probes possess unique optical properties and they can be used for biochemical studies for screening and characterization of protein kinase inhibitors as drug candidates [Enkvist E et al. ACS Chem Biol 201 1 ;6: 1052-1062; Lavogina D et al. Bioorg Med Chem Lett. 2012;22:3425-3430; Kasari M et al. Biochim Biophys Acta. 2013 Mar 14.

doi:pii:S 1570-9639(13)001 10-6.], for monitoring and mapping of protein kinase activity in living cells with microscopy [Vaasa A et al. Chem Commun (Camb). 2012;48:8595-8597], and determination of activity of protein kinases in biological samples (blood plasma, blood cells, tissue extracts, etc.) as biomarkers for diagnosis of diseases like cancer, atherosclerosis, diabetes, Alzheimer's disease, etc. [Kasari M et al. Anal Biochem. 2012;422:79-88].

The invention provides a compound of formula (I) containing a fluorophore as a

photoluminescent probe in an assay for measuring or monitoring the activity of a Pirn kinase. Optionally the assay is a competition assay and includes a Pirn kinase inhibitor, for example, with a view to determining the binding affinity of the inhibitor. The assay may be set up in a array format for determining the binding affinity of an array of inhibitors.

In an embodiment, the emission lifetime of the compound of formula (I) containing a fluorphore is 1-1000 microseconds when the compound is bound to a Pirn kinase.

The invention provides a binding assay for monitoring the activity of a Pirn kinase wherein the luminescence of a Pirn protein kinase complex with a compound of formula (I) containing a fluorophore is measured by time delayed (time-resolved) luminescence detection following pulse excitation. The binding assay may be performed on living cells. The binding assay may be an in vitro or ex vivo assay. Luminescence may be detected using a fluorescence microscope, a fluorescence spectrometer or a fluorescence platereader.

The invention also provides a method for performing a binding assay for monitoring the activity of a Pirn kinase which comprises time-delayed measurement of the luminescence of a Pirn protein kinase complex with a compound of formula (I) containing a fluorphore following pulse excitation.

Further details of such binding assays may be gleaned by reference to EP2482072 the contents of which are herein incorporated by reference in their entirety.

Salts and solvates

Compounds of formula (I) may be employed as salts, for example pharmaceutically acceptable salts. Salts of acids include salts formed with Group 1 and Group 2 metals (eg sodium, potassium, calcium, magnesium) and salts formed with ammonium ions. Salts of bases include acid addition salts, for example salts formed with mineral acids such as HC1, HBr, H₂SO₄ and organic acids such as acetic, succinic, fu marie acid, methane sulfonic acid, p-toluene sulfonic acid etc.

Compounds of formula (I) or their salts may be employed as solvates, such as hydrates.

Other embodiments

The compounds described herein may include one or more chiral centers, and, except where stated, the disclosure extends to include racemates, enantiomers and stereoisomers resulting therefrom. In one embodiment one enantiomeric form is present in a substantially purified form that is substantially free of the corresponding enantiomeric form.

The invention also extends to all polymorphic forms of the compounds of formula (I).

The invention also extends to isotopically-labelled compounds of formula (I) in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, fluorine, such as ²H, ³H, ⁿC, ¹⁴C and ¹⁸F. Isotopically labelled compounds of formula (I) may be prepared by carrying out the synthetic methods described below and substituting an isotopically labelled reagent or intermediate for a non-isotopically labelled reagent or intermediate.

The invention extends to all tautomeric forms of the compounds illustrated herein (particularly enol-keto tautomers). For example whereas formula (I) illustrates in some embodiments an enol form, the corresponding keto form is also embraced as part of the invention. The same applies to other structures herein which illustrate enol or keto forms of compounds.

Advantages

Compounds of formula (I) may have one or more of the following advantages as compared with prior art compounds:

-they are more potent as inhibitors of Pirn kinases;

-they are more selective as inhibitors of Pirn kinases; -they demonstrate good cell penetration ability;

-when they contain a fluorophore they exhibit photo luminescence following excitation with long lifetime.

Preparative methods

A general synthetic method for compounds of formula (I) incorporating a Q moiety of formula (Al) is given in Scheme 1 below:

Scheme 1. Synthesis of conjugates of tricyclic compounds and peptides

C I

Table 1. Structures of conjugates synthesized according to Scheme 1

A solution of 2-(chloromethyl)-thieno[2,3-d]pyrimidin-4(3H)-one (5 equiv.) in DMF and Fmoc- deprotected amino group of the corresponding peptide on the resin (1 equiv.) was treated with DIPEA at 80°C for 8 h in a flask with slow agitation. Completeness of the reaction was monitored with the Kaiser test and then resin was washed three times with DMF (2 ml), followed by cleavage from the resin with 2 to 3 h treatment with 90% trifluoroacetic acid (5% triisopropylsilane, 5% water). The peptide conjugate was purified by reversed phase HPLC (C I 8 column, water-CH₃CN gradient, 0.1% TFA). A general synthetic method for compounds of formula (I) incorporating a Q moiety of formula (C) is given in Scheme 2 below:

Scheme 2. Synthesis of conjugates of bicyclic compounds and peptides -Te the r -Peptd

Table 2. Structures of conjugates synthesized according to Scheme 2

The solution of substituted 3-amino-hetero-2-carboxylic acid in DMF was activated with the mixture of HBTU/HOBT (2.8 equiv. each) and N-methyl morpholine (9 equiv.), the resulting activated compound was added to Fmoc-deprotected peptide resin (1 equiv.) and agitated for 90 min. Completeness of the reaction was monitored with the Kaiser test. The product was cleaved from the resin with 2 to 3 h treatment with 90% trifluoroacetic acid (5% triisopropylsilane, 5% water). The peptide conjugate was purification by reversed phase HPLC (CI 8 column, water- CH₃CN gradient, 0.1% TFA).

Certain labeled compounds of formula (Q-L-P)-F may be prepared as follows.

Labeled compounds of formula (Q-L-P)-F in which F is coupled via a terminal lysine (e.g., D- Lys) residue may be prepared by reacting the compound of formula Q-L-P with a fluorophore provided with an activated carboxylic acid group (e.g., an acid activated by reaction with a carbodiimide or N-methyl succinimide) to give an amide bond connection.

Further methods of preparation may be gleaned from the Examples section.

Abbreviations

Pirn - 1-Provirus Integration site for Moloney murine leukemia virus

AGC - Protein kinase A, G, C

PKA - Protein kinase A

PKC - Protein kinase C

PKB - Protein kinase B

CAMK - calmodulin-dependent protein kinase CK2 - Casein kinase 2

B-CLL - B-cell chronic lymphocytic leukemia

MBHA - 4-methylbenzhydrylamine

K_D - Dissociation constant

nM - nanomolar

μΜ - micro molar

ARC - conjugate of a nucleoside analogue and an arginine-containing peptide

Ahx - 6-aminohexanoic acid

Aox - 8-aminooctanoic acid

Arg - arginine

Lys - lysine

Hyp - 4-hydroxy-L-proline

trans-Hyp - Trans-4-hydroxy-L-proline

DIPEA - N,N-diisopropylethylamine

DMSO - dimethyl sulfoxide

DMF - dimethyl formamide

HPLC - high performance liquid chromatography

HOBT - 1-hydroxybenzotriazole

Fmoc - 9-fluorenylmethylcarbonyl

MALDI-TOF MS - matrix assisted laser desorption ionization time-of- flight mass spectrometry

Pbf - 2,2,4,6, 7-pentamethyldihyrobenzofuran-5-sulfonyl

Boc - tert-butoxycarbonyl

RP - reverse phase

TFA - trifluoroacetic acid

TLC - thin layer chromatography

DTT - dithiothreitol

Examples

Synthetic examples

Peptide fragments were prepared by using traditional Fmoc solid phase peptide synthesis on Rink amide MBHA resin. In general procedure, protected amino acids (3 equiv.) were dissolved in DMF and activated with HBTU/HOBt (2.8 equiv. each) in DMF/N-methylmorpholine (9 equiv.). Coupling solutions were added to the resin and shaken for 1 h. The completeness of each step was monitored with the Kaiser test, which was followed by deprotection of Fmoc-group by 20 % piperidine solution in DMF in 2 min. for first time and same procedure followed for furthur 20 min. Fmoc protected linker (6-Fmoc-aminohexanoic acid, Fmoc-amino octanoic acid, and Fmoc-hydroxyproline methyl ester) was attached to the peptide part following the same protocol. The N-terminal Fmoc group was removed with 20% piperidine solution in DMF (20 min). The protection groups were removed and the conjugation cleaved from the resin with 2 h treatment with the solution containing 90% trifluoroacetic acid, 5% triisopropylsilane, 5% water. The conjugates were purified with C 18 reversed phase HPLC and lyophilized.

Example 1

Synthesis of ARC-3101

A solution of 2-(chloromethyl)-thieno[2,3-d]pyrimidin-4(3H)-one (5 equiv.) in DMF and Fmoc- deprotected amino group of the corresponding peptide on the resin (1 equiv.) was treated with DIPEA at 80 °C for 8 h in a round bottom flask with slow stirring. Completeness of the reaction was monitored with the Kaiser test and then resin was washed with three times with DMF (2ml), followed by cleavage from the resin with 2 to 3 h treatment with 90% trifluoroacetic acid (5% triisopropylsilane, 5% water). The peptide conjugate was purified by reversed phase HPLC (water-CH₃CN gradient, 0.1% TFA). MW (HRMS) = 1756.92. Example 2

Synthesis of ARC-3102

A solution of 3-amino-5-bromo-thiophene-2-carboxylic acid (3 equiv.) in DMF was activated with HBTU/HOBT (2.8 equiv. each) and N-methyl morpholine (9 equiv.) mixture. The resulting coupling solution was added to Fmoc-deprotected resin (1 equiv.) and agitated for 90 min.

Completeness of the reaction was monitored with Kaiser test and then followed by cleavage from the resin with 2 to 3 h treatment with 90% trifluoroacetic acid (5% triisopropylsilane, 5% water). The peptide conjugate was purification by reversed phase HPLC (water-CH₃CN gradient, 0.1 % TFA). MW (HRMS) = 1717.91.

Example 3

Synthesis of ARC-3104

A solution of 2-(chloromethyl)-thieno[2,3-d]pyrimidin-4(3H)-one (5 equiv.) in DMF and Fmoc- deprotected amino group of the corresponding peptide on the resin (1 equiv.) was treated with DIPEA at 80 °C for 8 h in a round bottom flask with slow stirring. Completeness of the reaction was monitored with the Kaiser test and then resin was washed with three times with DMF (2ml), followed by cleavage from the resin with 2 to 3 h treatment with 90% trifluoroacetic acid (5% triisopropylsilane, 5% water). The peptide conjugate was purified by reversed phase HPLC (water-CH₃CN gradient, 0.1% TFA). MW (HRMS) = 1601.17.

Examples 4

Synthesis of ARC -3106

The synthetic procedure for preparation of this peptide conjugate was similar to the procedure used for preparation of ARC-3102. MW (ESI-MS) = 1722.10. Example 5

Synthesis of ARC-3111

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3101. MW (ESI-MS) = 1759.14.

Example 6

Synthesis of ARC-3113

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3104. MW (ESI-MS) = 1601.17. Example 7

Synthesis of ARC-3114

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3102. MW (ESI-MS) = 1898.14.

Example 8

Synthesis of ARC-3115

1. HBTU/HOBT

2. TFA

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3102. MW (HRMS) = 1452.35.

Example 9

Synthesis of ARC-3116

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3104. MW (ESI-MS) = 1871.07. Example 10

Synthesis of ARC-3118

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3101. MW (HRMS) = 1672.77.

Example 11

Synthesis of ARC-3119

The synthetic procedure of this peptide conjugate was the same as used for the synthesis of ARC-3101. MW (HRMS) = 1942.11. Example 12

Synthesis of ARC-3120

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3101. MW (HRMS) = 848.81.

Example 13

Synthesis of ARC-3121

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3101. MW (HRMS) = 876.87. Example 14

Synthesis of ARC-3124

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3101. MW (HRMS) = 1161.18.

Example 15

Synthesis of ARC-3126

1. DI

2. TFA

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3101. MW (ESI-MS) = 1758.95.

Example 16

Synthesis of ARC-3127

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3102. MW (ESI-MS) = 1450.57.

Example 17

Synthesis of ARC-3157

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3104. MW (ESI-MS) = 1648.

Example 18

Synthesis of ARC-3160

The synthetic procedure of this peptide conjugate is similar to the procedure used synthesis of ARC-3104. MW (ESI-MS) = 2117.

Example 19

Synthesis of a fluorophore-labeled compound ARC-3108 from ARC-3104

Et₃N/DMSO

A solution of ARC-3104 (1.2 equiv.) in DMSO was treated with Promoflour-555 (1 equiv.) in the presence of excess triethylamine at room temperature for 3 h. After completion of the reaction, the solvent was removed in the freeze dryer and the product was purified by reversed phase HPLC (water-CH₃CN gradient, 0.1% TFA). MW (ESI-MS) = 2222.44. Example 20

Synthesis of a fluorophore-labeled compound ARC-3117 from ARC-3104

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3108. MW (ESI-MS) = 2248.48. Example 21

nthesis of a fluorophore-labeled compound ARC-3158 from ARC-3157

A solution of ARC-3157 (1.2 equiv.) in DMSO was treated with Promo fluor-647 (1 equiv.) in the presence of excess triethylamine at room temperature for 3 h. After completion of the reaction, the solvent was removed in the freeze dryer and the product was purified by reversed phase HPLC (water-CH₃CN gradient, 0.1% TFA). MW (ESI-MS) = 2272. Example 22

Synthesis of a fluorophore-labeled compound ARC-3159 from ARC-3157

A solution of ARC-3157 (1.2 equiv.) in DMSO was treated with PromoFluor-555 (1 equiv.) in the presence excess triethylamine at room temperature for 3 h. After completion of the reaction, the solvent was removed in the freeze dryer and the product was purified by reversed phase HPLC (water-CH₃CN gradient, 0.1% TFA). MW (ESI-MS) = 2245. Example 23

Synthesis of a fluorophore-labeled compound ARC-3161 from ARC-3160

A solution of ARC-3160 (1.2 equiv.) in DMSO was treated with Promofluor-647 (1 equiv.) in the presence excess triethylamine at room temperature for 3 h. After completion of the reaction, the solvent was removed in the freeze dryer and the product was purified by reversed phase HPLC (water-C¾CN gradient, 0.1% TFA). (ARC-3161, MW (ESI-MS) = 2739).

Examples 24-27

Intermediate compounds were prepared according to the following scheme:

n = 1-12

The mixture of tetrahalogenated benzimidazole (1 equiv.), the alkyl ester (n = 1 to 12, 1.2 equiv.) and .₂CO₃ (2 equiv.) was stirred in DMF at 60-70 °C for 3 to 4 h. Further the reaction mixture was extracted with ethyl acetate and evaporation of solvent provided the crude product, which was passed through a thin layer of silica gel using 3% MeOH in CHCI₃, leading to the product.

To a solution of tetrahalogenated N-alkylbenzimidazole ester in ethanol was added 4 M aqueous sodium hydroxide solution and the mixture was refluxed for 3 h. After removing of the solvents under vacuum, the residue was dissolved in water and then the product was precipitated with addition of 2 M aqueous hydrochloric acid solution to adjusted pH to 5. The resulting precipitate was filtrated, washed with water and dried under vacuum to give corresponding acid in good yield.

Synthesis of ARC-3125, ARC-3145, ARC-3146 and ARC-3150

The synthetic procedure for preparation of this peptide conjugate was similar to the procedure used for preparation of ARC-3102. MW (ESI-MS) = 1857.22.

The synthetic procedure for preparation of this peptide conjugate was similar to the procedure used for preparation of ARC-3102. MW (ESI-MS) = 1500.67

The synthetic procedure for preparation of this peptide conjugate was similar to the procedure used for preparation of ARC-3102. MW (ESI-MS) = 1184.30.

The synthetic procedure for preparation of this peptide conjugate was similar to the procedure used for preparation of ARC-3102. MW (ESI-MS) = 1528.67.

Examples 28-29

Intermediate compounds were prepared according to the following scheme:

X = Br, CI

n = 1-10

A mixture of tetrahalogenated phtha!ic anhydride (1 equiv.) and the respective amino acids (1 equiv.) in the presence of acetic acid was heated for 30 min with stirring in an oil bath at 140- 160 °C. After cooling, the solid material was washed with water, filtered and crystallized with ethanol. It yielded pale-yellow tetrahalogenated-N-phthaloy amino acid. Synthesis of ARC -3151 and ARC -3152

The synthetic procedure for preparation of this peptide conjugate was similar to the procedure used for preparation of ARC-3102. MW (ESI-MS) = 2155.56.

The synthetic procedure for preparation of this peptide conjugate was similar to the procedure used for preparation of ARC-3102. MW (ESI-MS) = 1886.21.

Example 30

Intermediate compounds were prepared according to the following scheme:

R¹= COOH, R²= H

Scheme 1

R¹= H, R²= COOH To the solution of diamine in 1M HCl was added selenium dioxide in water and the solution was stirred at 80°C until solution produced brown precipitate. The brown precipitate was filtered and dried. The product was used for the synthesis of peptide conjugates.

Synthesis -1601

The synthetic procedure for preparation of this peptide conjugate was similar to the procedure used for preparation of ARC-3102. MW (ESI-MS) = 1404.54.

Example 31

Preparation of a fluorophore-labeled compound ARC-1602 from ARC-1601

A solution of ARC-1601 (1.2 equiv.) in DMSO was treated with Promo fluor-647 (1 equiv.) in the presence excess triethylamine at room temperature for 3 h. After completion of the reaction, the solvent was removed in the freeze dryer and the product was purified by reversed phase HPLC (water-CH₃CN gradient, 0.1% TFA). MW (ESI-MS) = 2028.30.

Examples 32-35

Intermediate compounds were prepared according to the following scheme:

2. NaOH/Ethanol

reflux, 3hr

X = Se, Te

The mixture of 6H-[l ,2,5]selenachalcogendiazolo[3,4-e]indole (1 equiv.), bromo ethyl acetate (1.2 equiv.) and K CC (2 equiv.) was stirred in DMF at 60-70 °C for 3 to 4 h. Further the reaction mixture was extracted with ethyl acetate and evaporation of solvent provided the crude product, which was passed through a thin layer of silica gel using 20% ethyl, acetate in hexane to afford the desired product.

Synthesis of ARC -3200, ARC -3201 , ARC -3131 and ARC -3202

The synthetic procedure of this peptide conjugates is similar to the procedure used for the synthesis of ARC-3102. MW (ESI-MS) = (ARC-3200, MS 1471.63; ARC-3201 , MS 1520.27; ARC-3131 , MS 1471.63; ARC-3202, MS 1520.27). Example 36

Preparation of a fluorophore-labeled compound ARC-3132 from ARC-3331

A solution of ARC-3131 (1.2 equiv.) in DMSO was treated with Promofluor-647 (1 equiv.) in the presence excess triethylamine at room temperature for 3 h. After completion of the reaction, the solvent was removed in the freeze dryer and the product was purified by reversed phase HPLC (water-CH₃CN gradient, 0.1% TFA). MW (ESI-MS) = 2081.36.

Examples 37-38

Intermediate compounds were prepared according to the following scheme:

n =1 -10

A suspension of N-betizoylglycine (1 equiv.), sodium, acetate (1 equiv.), and acetic anhydride was stirred at room temperature for 30 min, thereafter benzo[c][ 1 ,2,5 jchalcogen diazole-5- carbaldehyde (1 equiv.) was added into the white suspension. The resulting suspension, was stirred at room, temperature for 1 h and then at 60 °C for 5 h. The reaction mixture became a bright solutio that upo cooling to room, temperature again became a suspension. This suspension was mixed with water and stirred at room, temperature for 30 min. The insoluble material, was separated by filtration, washed with water and recrystallized from methanol.

Amino acid (n = 1-10, 1.2 equiv.) was added to the solution of (4Z)-4- ((benzo[c][l,2,5]chalcogendiazol-6-yl)methylene)-2-phenyloxazol-5(4H)-one (1 equiv.) and K₂CO3 (2.2 equiv.) in ethanol. The reaction mixture was heated at reflux for 2 h, and then cooled to room temperature. After removal of solvent under reduced pressure, the product was purified by silica column chromatography to afford the corresponding product.

Synthesis of ARC-3203 and ARC3204

The synthetic procedure of this peptide conjugate (ARC-3203) was similar to the procedure used for the synthesis of ARC-3102. MW (ESI-MS) = 1858.08.

The synthetic procedure of this peptide conjugate (ARC-3204) was similar to the procedure used for the synthesis of ARC-3102. MW (ESI-MS) = 1906.72.

Examples 39

Intermediate compounds were prepared according to the following sche

Se

2. Reflux, 3h

COOCH2CH3

Et₃N, DMF reflux To the solution containing the corresponding malonic acid derivative (1 equiv.), piperidone (1 equiv.) and corresponding selenium or tellurium (3 equiv.) in DMF, was refiuxed in the presence of triethylamine for 6 to 8 h. When the reaction was completed the mixture was filtered with charcoal and poured onto the crushed ice. The crystals were filtered off and re-crystallized with ethanol. A suspension of ethyl 2-amino-4, 5, 6, 7-tetrahydrochalcogenpheno [3, 2- c]pyridine-3-carboxylate (1 equiv.) in 4 N hydrochloric acid in dioxane (5 mL) was treated with 2-chloroacetonitrile (3 equiv.) at room temperature for 3 h and then refiuxed for 2 h. The white solid was collected by filtration, washed with ethanol and dried to give the desired product in good yield.

Synthesis of ARC-3205

The following compounds were prepared using methods described herein:

The synthetic procedure of this peptide conjugate is similar to the procedure used for the synthesis of ARC-3104. MW (ESI-MS) = 1730.98.

Examples 40-42

Synthesis of ARC-1500, ARC-3206 and ARC-3207

The synthetic procedure of these peptide conjugates were similar to the procedure used for the synthesis of ARC-3102. MW (ESI-MS) = (ARC- 1500, MS 1687.06; ARC-3206, MS 1686.07; ARC-3207, MS 1714.08).

Example 43

Synthesis of myristoylated compound ARC-3222 from ARC-3104

A solution of ARC-3104 (1 equiv.) in DMSO was treated with myristic anhydride (1 equiv.) in the presence triethylamine (1.5 equiv.) at room temperature for 1 h. After completion of the reaction, the solvent was removed in the freeze dryer and the product was purified by reversed phase HPLC (water-CH₃CN gradient, 0.1% TFA). (ARC-3222, MW (ESI-MS) = 1775.00). Testing examples

Example 44 - Selectivity of Inhibitors

The selectivity testing for ARC-compounds ARC-3104, ARC-3125, ARC-3126 was performed in a panel of more than 120 kinases (Table 4). This testing revealed that these compounds possessed very high inhibitory potency to the kinases of the Pim family, whereas the general protein kinase inhibition profile strongly depended on the structure of the aromatic fragment Q and the tether L of the conjugate according to formula (I).

The results of the testing are expressed as the percentage of residual activity of the kinase in the presence of the inhibitors (ARC-3104 at 50 nM concentration, and ARC-3125, ARC-3126, both at 1 μΜ concentration). The selectivity profiling showed that ARC-3104, ARC-3125, ARC-3126 efficiently inhibited protein kinases Pim-1, Pim-2, Pim-3. ARC-3104 at 50 nM concentration caused 83% inhibition of Pim-1, 60% inhibition of Pim-2 and 69% inhibition of Pim-3. ARC- 3125 at 1 μΜ concentration caused 100% inhibition of Pim-1 and Pim-2 activity, and 93% inhibition of Pim-3 activity. ARC-3126 at 1 μΜ concentration caused 100% inhibition of Pim-1 activity, 90% inhibition of Pim-2 activity and 87% inhibition of Pim-3 activity. Both ARC-3104 and ARC-3126 revealed high selectivity for inhibition of Pim kinases compared to other protein kinases. ARC-3125 with very high potency inhibited Pim kinases and with high potency also inhibited several protein kinases belonging to other groups of protein kinases (PKBa, CLK2, PKBB, CAMKl, IGF-IR, ROCK2, MSKl, AMPK, PKA, NUAKI, SGKI, AMPK, TAKl, for all more than 90% inhibition).

Table 4. Selectivity profile of selected inhibitors of the present invention towards protein kinases. Residual activities (%) of protein kinases in the presence of inhibitors ARC-3125 (1 μΜ), ARC-3126 (1 μΜ), ARC-3104 (50 nM) are reported (the measurements were performed at the MRC Protein Phosphorylation Unit, Dundee, UK).

Src 153 103 85

ERK2 161 166 106

Lck 226 212 87

Example 45

Pim-1 binding of compounds of formula (I)

Table 5. Structures of probes ARC-3108 and ARC-3117 according to the general structure (Q-L- P)-F and a myristoylated peptide conjugate ARC-3122, and their affinity towards the protein kinase Pim-1. The values of the dissociation constant Ka were calculated from IC₅₀ values determined with the assay of Example 48 (ARC-3108 and ARC-3117) or Example 46 (ARC- 3122)

Example 46

Determination of the affinity of an inhibitor with ARC-Lum-based binding/displacement assay

The Ka values of inhibitors were determined using a displacement assay as described before [Enkvist, E. et al 2011, ACS Chem Biol, 6(10), 1052-1062]. The concentration series of inhibitors (3-fold dilutions) were made in the assay buffer (150 mM NaCl, 50 mM Hepes pH = 7.5, 5 mM dithiothreitol, 0.5 mg/mL bovine serum albumin, 0.005% Tween-20), and a mixture of Pim-1 kinase (final concentration 3 nM) in complex with a luminescent probe ARC- 1139 (final concentration 100 nM) was added to each well (final volume 20 μί). The microplates were incubated for 20 min at 30 °C and the intensity of protein induced luminescence arising from the remaining complexes of ARC- 1139 and Pim-1 was quantified on a PHERAstar microplate reader (BMG Labtech) with TRF optical module [excitation at 337 (50) nm, emission 675 (50)] using the time-resolved fluorescence measurement mode. Samples were excited with a flash of the xenon lamp (200 flashes per data point) at 337 nm, followed by delay time of 50 and acquisition time of 150 μβ. Luminescence intensity was plotted against the logarithm of the concentration of the inhibitor (Figure 1) and the obtained displacement curves were fitted to a sigmoidal dose response model to obtain IC₅₀ values that were recalculated into the values of dissociation constant Ka which are listed in Table 6.

Table 6. Affinities (Ka values) of some ARC-compounds towards Pim-1 kinase

Compound Ka,* nM Compound Ka,* nM

ARC-3102 1.2 ARC-3113 1.1 ARC-3106 0.5 ARC-3116 0.6

ARC-3115 1.9 ARC-3124 20

ARC-3127 1.9 ARC-3118 0.6

ARC-3101 < 0.1 ARC-3119 0.4

ARC-3111 0.4 ARC-3120 189

ARC-3126 0.3 ARC-3121 227

ARC-3104 0.4 SGI- 1776 4.8

Staurosporine 5.2 (D-Arg)₉-NH₂ 290

^Values of displacement constants Kj in brackets were calculated according to the Cheng- Prusoff equation [Enkvist E. et al 2011, ACS Chem Biol, 6(10), 1052-1062].

Example 47

Omnia Kinase Assay (with Life Technologies Corporation kit)

The inhibition IC₅₀ values of inhibitors towards protein kinase Pim-1 were measured according to the Omnia Kinase Assay protocol (Life Technologies Corporation). All inhibition experiments were performed on black low volume 384-well non-bonding-surface microplates (code 3676, Corning) on a PHERAstar platereader (BMG Labtech). The concentration series of inhibitors (3 -fold dilution) were made in assay (Kinase Reaction Buffer) buffer. Each dilution in the series was at 2X of the final concentration of inhibitor in the reaction. Thereafter 5 μΐ of the 4X mixture of ATP (4 mM), DTT (0.8 mM) and Omnia Peptide Substrate ST26 (40 μΜ) were added to each well. Plate was incubated for 5 minutes at 30 °C. Reactions were initiated by addition of 5 μΐ of 4X (20 nM) kinase to the reaction mixtures and fluorescence intensity readings (ex. 360 nm, em. 485 nm) were recorded every 30 seconds for 60 minutes. The initial velocities of each reactions were determined (the slope of a plot of relative fluorescence units vs time) and plotted against the inhibitor concentration (Figure 2). To obtain IC₅₀ values the curves were fitted to a sigmoidal dose response model with the aid of GraphPad Prism 5 software (GraphPad Software, Inc.). The values of the inhibition constant for some of the inhibitors that are listed in Table 7 are close to the values of dissociation constants Kj (Table 6) characterizing the affinity of inhibitors to Pim-1 kinase.

Table 7. Values of inhibition constant (as determined with Omnia Kinase Assay) for selected inhibitors towards Pim-1

Compound K (nM)

ARC-3102 K = 1.4 + 0.6 nM

ARC-3104 K < 0.5 nM ARC-3120 ¾ = 145 ± 45 nM

SGI- 1776 Ki = 4.5 ± 1.5 nM

Example 48

Titration of a responsive ARC-Lum probe ARC-3158. Assay for determination of affinity of fluorophore-labeled probes

Embodiments of the invention include compounds that possess microsecond-scale emission life time photoluminescence properties when bound to protein kinases. Free probes are devoid of these properties. Thus such probes are responsive probes that can be used for monitoring and mapping of Pirn kinase activity in living cells [Vaasa A et al. Chem Comrnun (Camb). 2012;48:8595-8597]. The physical mechanism behind the optical phenomenon has been described for structurally different conjugates by authors of the present invention [EP2482072; Enkvist E et al. ACS Chem Biol 201 1;6: 1052-1062]. The probes disclosed in these publications revealed very weak long lifetime emission signal in complex with Pirn kinases that was not sufficient for practical applications. Thus novel probes disclosed in the present invention (ARC- 3158, 3159, 3161, ARC-1602) with high affinity bind to Pirn kinases and the probe/Pim-1 complex emits phosphorescence with microsecond lifetime in 500 - 650 nm region from the complex is excited with a pulse of light in UV region (between 200 - 400 nm). Compounds labelled at free amino acid residue with a fluorophore absorbing light in 450 - 700 nm range possess amplification of the emission signal of the phosphorescence donor and emit light at wavelengths of emission spectrum of the acceptor fluorophore.

Here the responsive probe ARC-3158 comprising a selenium-containing ring and PromoFluor 647 dye (2 nM) was titrated with Pim-1 kinase.

The biochemical binding experiments were performed on black low volume 384-well non- bonding-surface microplates (code 3676, Corning) on a PHERAstar platereader (BMG Labtech) with TRF optical modules [ex. 337(50) nm, em. 675(50) nm] using the time-resolved

fluorescence measurement mode. The microplates were incubated at 30°C for 15 min before each measurement.

To characterize the binding of ARC-3158 to Pim-1 the concentration series of the kinase (3-fold dilutions) was made in the assay buffer and the fixed concentration of luminescent probe ARC- 3158 (final concentration 2 nM) was added to each well.

In TRF mode, ARC-Lum probes were excited with a flash of the xenon lamp at 337(50) nm, followed by 50 delay time and subsequent acquisition (150 μβ) of the luminescence at 675(50) nm.

The data from the assays were fitted with the aid of GraphPad Prism software version 5.0 (GraphPad Software, Inc.) (see Figure 3) and the K_D value of 0.5 nM was calculated using nonlinear regression analysis. Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

All patents and patent applications referred to herein are incorporated by reference in their entirety.

Claims

Claims

1. A compound of formula (I),

Q-L-P (I)

wherein:

Q is a moiety that binds to the ATP -binding site of a Pirn kinase selected from any one of structures A to J:

wherein either U represents H and V represents (K)p-Pv4 or U represents CH₂(K)p-Pv4 and V represents (CH₂)zJe;

z represents 0 or 1 ;

Je represents a 5-7 membered heterocyclic ring containing 1 or 2 heteroatoms selected from O, N and S; and

in the case of when V is (CH₂)zJe and U is CH₂(K)p-Pv4, said moiety of formula (A) being optionally substituted on one or more of the aromatic rings by one or more halogen groups;

(B)

wherein AEF represents N(CH₂(K)P-P )-CH=CH or CH=CH-N(CH₂(K)P-P );

said moiety of formula (B) being optionally substituted on one or more of the aromatic rings by one or more halogen groups;

said moiety of formula (D) being optionally substituted on the phenyl ring by one or more (e.g. one) halogen groups;

wherein Z represents CH or N; and

said moiety of formula (E) being optionally substituted on one or both aromatic rings by one or more groups selected from halogen, Cl-4alkyl or -CHO;

said moiety of formula (F) being optionally substituted on the phenyl ring by one or more halogen groups;

(G)

wherein Ph represents phenyl which may optionally be substituted by one or more substituents selected from Cl-4alkyl and halogen; and

said moiety of formula (G) being optionally substituted on one or more of the aromatic rings by one or more halogen groups;

(H)

wherein W represents CH₂ or NH; and

said moiety of formula (J) being optionally substituted on one or more of the aromatic rings by one or more halogen groups;

Ri represents Br, CI, I, or H; P 2 represents Br, CI, I, H, or OH;

P 3 represents CH or N;

P4 represents the point of attachment to L-P;

Rs represents O, S, Se, Te, NH, N-CH₃;

K represents a link;

p represents 0 or 1 ;

X represents Se or Te;

L is an organic tether that connects Q to P and permits simultaneous binding of Q and P to a Pim kinase;

P is a moiety that binds to the protein binding domain of Pim kinase; and

wherein the compound of formula ( I ) is optionally labelled with a fluorophore.

2. A compound of formula (I) according to claim 1 wherein Q represents a moiety of structure (A):

A compound of formula (I) according to claim 2 wherein Q represents a moiety of structure (Al):

A compound of formula (I) according to claim 3,

wherein

K represents -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

q represents 0 or an integer 1-6; and

r represents 0 or an integer 1-6.

A compound of formula (I) according to claim 1 wherein Q represents a moiety of structure (Bl) or (B2):

6. A compound of formula (I) according to claim 4,

wherein

K represents -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

q represents an integer 1-6; and

r represents 0 or an integer 1-6.

7. A compound of formula (I) according to claim 1 wherein Q represents a moiety of structure (C):

(C)

8. A compound of formula (I) according to claim 1 wherein Q represents a moiety selected from one of structures D to H:

wherein

K represents -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)- q represents an integer 1-6; and

r represents 0 or an integer 1-11;

wherein

K represents -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

q represents 0 or an integer 1-6, save when Z is N in which case it has a minimum value of 1 ; and

r represents 0 or an integer 1-6;

wherein

K represents -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

q represents an integer 1-6; and

r represents 0 or an integer 1-11;

wherein

K represents -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

q represents an integer 1-6; and

r represents 0 or an integer 1-6; and

wherein

K represents -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

q represents 0 or an integer 1-6; and

r represents 0 or an integer 1-6.

A compound of formula (I) according to claim 1 wherein Q represents a moiety of structure (J):

(J)

wherein

K represents -(CH₂)_q-OC(0)- or -(CH₂)_r-C(0)-;

q represents an integer 1-6; and

r represents 0 or an integer 1-6.

10. A compound of formula (I) according to any one of claims 1 to 3 wherein R5 represents S.

11. A compound of formula (I) according to any one of claims 1 to 3, 7 and 10 wherein Ri represents H.

12. A compound of formula (I) according to any one of claims 1 to 3, 7, 10 and 11 wherein R₂ represents Br.

13. A compound of formula (I) according to any one of claims 1 to 3, 7 and 10 to 12 wherein R3 represents CH.

14. A compound of formula (I) according to any one of claims 1 to 13 wherein P represents a peptidic fragment incorporating one or more arginine residues or a peptidomimetic incorporating one or more guanidine groups.

15. A compound of formula (I) according to claim 14 wherein P includes two or more D- amino acid residues.

16. A compound of formula (I) according to claim 15 wherein P includes a (D-Arg)_n moiety wherein n is 2-10.

17. A compound of formula (I) according to claim 14 wherein P includes two or more

guanidine groups.

18. A compound of formula (I) according to claim 14 wherein P includes a terminal Lys (e.g.

D-Lys) residue.

19. A compound of formula (I) according to claim 18 wherein myristic acid or another

hydrophobic fatty acid is attached to the free amino group of a lysine residue in the P moiety.

20. A compound of formula (I) according to any one of claims 16, 18 or 19 wherein P

includes a (D-Arg)₆-(D-Lys)- moiety.

21. A compound of formula (I) according to any one of claims 1 to 20 wherein L includes the residue of the amino acid H₂N-(CH₂)_m-COOH in which m represents an integer 3-10, preferably 6-8.

22. A compound of formula (I) according to any one of claims 1 to 21 wherein L includes a Hyp residue, especially trans-Hyp.

23. A compound of formula (I) according to any one of claims 1 to 21 wherein L includes an Ahx moiety.

24. A compound of formula (I) according to any one of claims 1 to 21 wherein L includes a moiety selected from Hyp, Hyp-Ahx and Hyp-Aox e.g. where each Hyp is trans-Hyp.

25. A compound of formula (I) according to any one of claims 1 to 24 wherein the Pim kinase is Pim-1 kinase.

62

63

64

65

66

68

and salts of any one thereof.

27. A compound of formula (I),

Q-L-P (I) wherein

Q is a moiety that binds to the ATP -binding site of a Pirn kinase;

L is an organic tether that connects Q to P and permits simultaneous binding of Q and P to a Pirn kinase;

P is a moiety that binds to the protein binding domain of Pirn kinase; and

wherein the compound of formula (I) is optionally labelled with a fluorophore.

28. A compound of formula (I) according to claim 27 wherein Q represents an aromatic or heteroaromatic ring system comprising a fused bicycle or tricycle for example is a fused 5-6 fused ring system or a 6-5-6 fused ring system.

29. A compound of formula (I) according to any one of claims 1 to 28 wherein Q, L or P incorporate a fluorophore.

30. A compound of formula ( I ) according to claim 29 wherein P incorporates a fluorophore.

31. A compound of formula ( I ) according to claims 30 wherein the fluorophore is

incorporated via coupling to a D-Lys residue in P.

32. A compound of formula (I) according to any one of claims 29 to 31 wherein the

fluorophore is PromoFluor 647 or PromoFluor 555.

and salts of any one thereof.

A compound of formula (I) according to claim 30 which is:

or a salt thereof.

35. A compound of formula (I) according to any one of claims 1 to 26 for use as a

pharmaceutical.

36. A compound of formula (I) according to any one of claims 1 to 26 for use in the treatment or prevention of a condition associated with activity of a Pirn kinase.

37. A compound of formula (I) according to claim 36 wherein the condition is cancer.

38. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1 to 26 and one or more pharmaceutically acceptable diluents or carriers.

39. A method of treatment or prevention of a condition associated with activity of a Pirn

kinase which comprises administering to a patient in need thereof a pharmaceutically effective amount of a compound of formula (I) according to any one of claims 1 to 26.

40. Use of a compound of formula (I) according to any one of claims 29 to 34 in an assay for determining the activity or amount of a Pirn kinase inhibitor.

41. Use of a compound of formula (I) containing a fluorophore according to any one of

claims 29 to 34 as a photoluminescent probe in an assay for measuring or monitoring the activity of a Pirn kinase.

42. Use of the compound of formula (I) according to any one of claims 29 to 34 in an assay for the identification of a Pirn kinase inhibitor by its displacement of a compound of formula (I) of any one of claims 29 to 34 from its complex with the Pirn kinase by an inhibitor compound present in the solution comprising the steps of:

1) optionally establishing the Kj for the bound complex of the compound of formula (I) incorporating a fluorophore with the Pirn kinase;

2) contacting the compound of formula (I) incorporating a fluorophore with the Pirn kinase and measuring the fluorescence signal of the formed complex;

3) incubation of the complex formed in the previous step with the potential inhibitory compound or a mi ture of compounds and measuring of the fluorescence signal of the compound of formula (I) incorporating a fluorophore in its complex with the Pirn. kinase; 4) comparing the fluorescence signals from step 2) and step 3), a reduction in the fluorescence signal o step 3) as compared with that of step 2) indicating that the potential inhibitory compound or a mixture of compounds is capable of binding to the Pim kinase.

A binding assay for monitoring the activity of a Pim kinase wherein the luminescence of a Pim protein kinase complex with a compound of formula (I) according to any one of claims 29 to 34 is measured by time delayed (time-resolved) luminescence detection following pulse excitation.