SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 12, 2021, is named VPH-01001_SL.txt and is 599 bytes in size.

The present invention relates to a compound of formula (I)


wherein
X is C═O, C═S or B—OH;
Y is an electrophile and Z is a leaving group, or Y═Z is an electrophile;
R1comprises or consists of(a) (i) a first group binding to a proteolytic site of a proteasome, said first group being bound to X; and(ii) optionally a second group enhancing delivery;or(b) a group binding between subunits β1 and β2 of a proteasome;
R2and R3are independently selected from H, methyl, methoxy, ethyl, ethenyl, ethinyl and cyano, wherein methyl and ethyl may be substituted with OH or halogen.

In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

In recent years, the proteasome has been validated as a therapeutic target for anti-cancer therapy and the first proteasome inhibitor Bortezomib has been approved in 2008 for the treatment of multiple myeloma and mantle cell lymphoma. Current efforts focus on the development of second generation inhibitors with improved pharmacological properties. Epoxyketone inhibitors of the proteasome are desirable cancer therapeutics. The dose required to elicit inhibition is considerably lower than the boronic acid proteasome inhibitor Bortezomib. Additionally, many patients treated with Bortezomib acquire resistance to this agent through mutations in the β-subunits, i.e. the active site subunits of the proteasome. It has been recognized in recent years that Bortezomib resistant patients are responsive to Carfilzomib, which is the only epoxyketone inhibitor approved for clinical use today. This further highlights the importance of the development of additional epoxyketone inhibitors and/or inhibitors exhibiting similar inhibition properties as epoxyketone inhibitors. In pursuit of this goal most approaches today focus on the modification of the peptide backbone of epoxyketone inhibitors. These modifications include the introduction of natural and non-natural amino acids to increase the specificity for a given proteasome active site, the solubility of epoxyketone inhibitors, the bioavailability and the possibility to orally administer cancer therapeutics. In addition, medicinal chemistry approaches have focused on the addition of capping agents at the S4 position, which increase the absorptive properties of epoxyketone inhibitors and/or protect them from the harshly acidic environment of the gastro-intestinal tract.

The parent molecule of this class epoxyketone proteasome inhibitors, epoxomicin, was first isolated as a natural product synthesized by anActinomycesbacterial strain. Later, another molecule of this class was found, viz. eponemycin, and several synthetic variants of these molecules (FIG. 1) are presently in clinical trials as cancer therapeutics (Bennett and Kirk, Curr. Opin. Drug Discov. Devel., 11, 616-625 (2008); Demo et al., Cancer Res., 67, 6383-6391 (2007)).

The efficacy of epoxyketone inhibitors, is a consequence of the dual electrophile nature of the epoxyketone group. Based on several co-crystal structures of epoxyketone inhibitors with yeast, mouse and human 20S proteasomes, it has been described that the γ-hydroxyl group of the proteasome active site catalytic threonine reacts with the ketone moiety, whereas the N-terminal amino group of Thr1 reacts with the carbon atom of the epoxide of the inhibitor, which is in α-position to the ketone, in an irreversible manner. As shown in Scheme 1, this suggested chemistry results in the formation of an 1,4-morpholine ring product, which is formed by the active site catalytic threonine and the epoxyketone inhibitor (Groll et al., Journal of the American Chemical Society, 122, 1237-1238 (2000)).



This proposed inhibition scheme was formulated based on crystal structures where the electron density in the 20S proteasome active site was of insufficient resolution and quality to unequivocally model the inhibited state in atomic detail. Yet, the common belief that irreversible proteasome inhibition by epoxyketones yields a six atom ring adduct is well-established in the art; see, for example, the section dedicated to Carfilzomib and references cited therein in the review about proteasome inhibitors by Kubiczkova et al. (J. Cell. Mol. Med., 18, 947-961 (2014)).

There remains a significant need to develop new proteasome inhibitors, in particular improved proteasome inhibitors, improvements including activity at lower dosage and less side effects.

This technical problem has been solved by the subject-matter of the enclosed claims.

Accordingly, the present invention, in a first aspect relates to a compound of formula (I)


wherein
X is C═O, C═S or B—OH;
Y is an electrophile and Z is a leaving group, or Y═Z is an electrophile;
R1comprises or consists of(a) (i) a first group binding to a proteolytic site of a proteasome, said first group being bound to X; and(ii) optionally a second group enhancing delivery;or(b) a group binding between subunits β1 and β2 of a proteasome;
R2and R3are independently selected from H, methyl, methoxy, ethyl, ethenyl, ethinyl and cyano, wherein methyl and ethyl may be substituted with OH or halogen.

It is understood that throughout the present specification the term “compound” encompasses pharmaceutically acceptable salts, solvates, polymorphs, prodrugs, codrugs, cocrystals, tautomers, racemates, enantiomers, or diastereomers or mixtures thereof unless mentioned otherwise.

The compounds of formula (I) have a bipartite structure. R1, herein also referred to as “targeting group”, is responsible for targeting the compound of formula (I) to the proteolytic site of the proteasome. This part of the compound may build on established knowledge; for details see below. The remainder of said compound is displayed in more detail in formula (I). This part is also referred to as “headgroup” or “warhead” herein. It is responsible for the irreversible inhibition of the proteasome. The headgroup requires an electrophile (Y or Y═Z) in β-position relative to the functional group X. Such a design is contrary to the common belief that irreversible inhibition of the proteolytic site of the proteasome yields a morpholine ring. To explain further, as can be seen in Scheme 1 displayed herein above, the expected mechanism involves a nucleophilic attack of the amino group of Thr1 on a position which is in a to the ketone of the epoxyketone inhibitor. The epoxy group is amenable to such nucleophilic attack, noting that the carbon in α-position to the ketone is electrophile.

The present inventors determined more than 20 crystal structures of the human 20S proteasome at improved resolutions ranging from 1.8-2.2 Å. These new crystal structures of human 20S proteasomes provide insight into the inhibited state at atomic resolution. In contrast to the previously described formation of a 6-membered 1,4-morpholine ring structure for epoxyketone proteasome inhibitors, it is now possible to visualize the formation of a 7-membered 1,4-oxazepane ring structure involving the catalytic threonine residue of the proteasome active site and the inhibitor in the inhibited state. Formation of such an 1,4-oxazepane inhibited state is achieved by a double electrophilic reactive group on the inhibitor, where both electrophiles are in a distance of two atoms to one and another.

This finding allows to propose a novel chemical mechanism for the inhibition of 20S proteasomes by epoxyketone inhibitors. In this reaction mechanism, initially the γ-hydroxyl group of the proteasome active site catalytic threonine reacts with the ketone moiety, similar to the situation where the 1,4-morpholine ring (6-ring) structure is formed. In contrast to established theory, and to allow for 1,4-oxazepane 7-ring formation, the N-terminal amino group of Thr1 reacts in an irreversible manner with that carbon atom of the epoxide of the inhibitor which is in β-position to the ketone (Scheme 2). The reaction with the carbon atom of the epoxide in β-position to the ketone appears to be favored because it is the less substituted center.



From a theoretical perspective, epoxyketone inhibitors are capable of forming either type of adduct, namely a 6-ring or a 7-ring. However, the formation of a 7-ring adduct has never been considered. Instead, the prior art is consistent in that a morpholine ring would be formed. The present inventors' work is first to establish that a 7-ring adduct is formed. As a consequence, the present inventors are the first to disclose the specific molecular architecture as laid down in formula (I) which is specifically tailored for the formation of a 7-ring adduct. The inhibitory activity of the art-established epoxyketones entirely fails to suggest this mechanism. Only in view of the high resolution crystal structures, the present inventors were able to discover the β-regiospecificity of the nucleophilic attack of the active site threonine residue of the proteasome.

Without wishing to be bound to a specific theory, the present inventors furthermore consider that the formation of a seven ring adduct is kinetically favoured. Therefore, the provision of the compounds of the first aspect of the present invention which are specifically tailored for said kinetically favoured reaction mechanism will allow the use of lower dosages. As a consequence, fewer side effects are expected.

X is a functional group providing an electrophile. It can be a keto group, a thio-keto group or a boronic acid group. Deviant from the boronic acid group as present, for example, in Bortezomib, the compound of formula (I) requires that boron is part of the main chain. As shown in Scheme 2, the oxygen of the hydroxy group of the active site threonine residue of the proteasome binds to the electrophile atom in group X (the carbon atom or the boron atom).

The group Y—Z defines a second electrophile. Either Y as such or Y—Z as a whole is electrophile; for preferred implementations see further below.

Moieties R2and R3define the intervening portion of the compound between the two electrophiles.

R1is the above mentioned targeting moiety. Targeting moieties are known in the art. In many instances, targeting moieties are peptidic in nature. This applies, for example, to Epoxomicin, Oprozomib, Carfilzomib, Dihydroeponomycin, Eponemycin and ONX-0914. Since the active site of the proteasome exhibits proteolytic activity, peptides generally bind to said active site and are accordingly useful for targeting.

Said first group may optionally be bound to a second group which second group enhances delivery. To the extent the first group is peptidic in nature and consists of three amino acids or derivatives thereof, said second group may occupy a fourth position, in the art also referred to as S4 position. Dorsey et al. (J. Med. Chem., 51, 1068-1072 (2008)) and Zhou et al. (J. Med. Chem., 52, 3028-3038 (2009)) explored several suitable groups which are attached to the N-terminal end of a peptidic first group. These groups attached to the N-terminal end of a peptidic first group as described in these two publications all constitute suitable second groups in accordance with the present invention.

An alternative term for said second group is “N-cap”.

While peptidic first groups are envisaged, the particular structure of the targeting group is not key to the present invention. Basically, any chemical group which provides for targeting to a proteolytic site in the proteasome or to the vicinity thereof is a suitable group R1in accordance with the present invention. To the extent a targeting group does not target directly to the proteolytic site, but to another site of the proteasome in the vicinity of the proteolytic site, linkers of suitable length may be used to connect the targeting group R1to the active element of the compound of the first aspect, said active element comprising groups X, Y and Z.

To give an example of alternative targeting groups, Beck et al. (Angew. Chem. Int. Ed. 54, 11275-11278 (2015)) describe a molecule binding to the crevice between β1 and β2 subunit of the proteasome. This compound is displayed below.



Replacing the ethyl group of the ethoxy moiety bound to the phenyl group of said compound is a means of rendering this molecule suitable group R1. This is explained in further detail below.

The term “pharmaceutically acceptable salt” refers to a salt of a compound of the present invention. Suitable pharmaceutically acceptable salts include acid addition salts which may, for example, be formed by mixing a solution of compounds of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compound carries an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrative examples of pharmaceutically acceptable salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like (see, for example, S. M. Berge et al., “Pharmaceutical Salts”, J. Pharm. Sci., 66, pp. 1-19 (1977)).

When the compounds of the present invention are provided in crystalline form, the structure can contain solvent molecules. The solvents are typically pharmaceutically acceptable solvents and include, among others, water (hydrates) or organic solvents. Examples of possible solvates include ethanolates and iso-propanolates.

The term “codrug” refers to two or more therapeutic compounds bonded via a covalent chemical bond. A detailed definition can be found, e.g., in N. Das et al., European Journal of Pharmaceutical Sciences, 41, 2010, 571-588.

The term “cocrystal” refers to a multiple component crystal in which all components are solid under ambient conditions when in their pure form. These components co-exist as a stoichiometric or non-stoichometric ratio of a target molecule or ion (i.e., compound of the present invention) and one or more neutral molecular cocrystal formers. A detailed discussion can be found, for example, in Ning Shan et al., Drug Discovery Today, 13(9/10), 2008, 440-446 and in D. J. Good et al., Cryst. Growth Des., 9(5), 2009, 2252-2264.

The compounds of the present invention can also be provided in the form of a prodrug, namely a compound which is metabolized in vivo to the active metabolite. Suitable prodrugs are, for instance, esters. Specific examples of suitable groups are given, among others, in US 2007/0072831 in paragraphs [0082] to [0118] under the headings prodrugs and protecting groups.

To the extent compounds of the invention exhibit a pH-dependent charged state, it is understood that all possible charged states are embraced. A preferred pH range in this regard is from 0 to 14.

To the extent a compound according to the invention bears a net charge, it is understood that the compound is provided in electroneutral form. This is achieved by one or more counterions, preferred counterions being defined in relation to the term “salt” herein above.

In a preferred embodiment, said first group is a peptidic group comprising or consisting of at least two amino acids, or a corresponding peptidomimetic, wherein X takes the place of the carbonyl group or of the α-carbon of the C-terminal amino acid of said peptidic group, and said second group, if present, is bound to the N-terminus of said peptidic group.

Preferred amino acids are α-amino acids. Preferred α-amino acids are the 20 proteinogenic amino acids. Also preferred are derivatives of said proteinogenic amino acids, for example, the methyl ester of serine, also denoted “Ser(OMe)”. Other derivatives of the proteinogenic amino acids may be used as well, for example those described in Zhou et al. loc. cit. and including 4-diazolyl-alanine, homoserine methyl ester, pyridyl alanines such as 2-pyridyl alanine, 3-pyridyl alanine and 4-pyridyl alanine; 3-thienyl alanine, cyclohexyl alanine, cyano alanine and methyl serine.

Also other α-amino acids such as 2-amino butyric acid may be used.

In further preferred embodiments, β-amino acids may be used in one or more positions of the peptidic group. Also preferred is to use D-amino acids at one or more positions.

As an alternative to peptide bonds, alternative functional groups may be used. This may apply to a single peptide bond or to two or more peptide bonds. If all peptide bonds are replaced by other functional groups, the backbone chemistry in its entirety is changed. Alternative backbones are known in the art and include polyactide (PLA), alkylamines, jeffamines and those shown below ((a) to (r)) and described in Grimm et al. (Acta Cryst D (2010), 66, 685-697).



The above displayed backbones are as follows.

(a) M-type Jeffamines. R1═—H for ethylene oxide (EO) or —CH3for propylene oxide (PO). The PO/EO molar ratio is 29/6 for Jeffamine M2005, 10/31 for Jeffamine M2070 and 9/1 for Jeffamine M600. (b) Pentaerythritol ethoxylate. (c) Pentaerythritol propoxylate. (d) Polyvinyl pyrrolidone. (e) Polypropylene glycol. (f) Polyvinyl alcohol. (g) Polyacrylate. (h) Cellulose-based polymers. R1═—H, —CH3or —CH2CHOHCH3(hydroxypropyl methylcellulose), —H or CH2CO2H (carboxymethyl cellulose). (i) Poly(ethylene imine). (j) Di[poly(ethyleneglycol)]adipate. (k) Jeffamine ED2003. (l) Jeffamine D2000. (m) Jeffamine SD2001. (n) T-type Jeffamines. (o) Polyacryl amide. (p) Glycerol ethoxylate. (q) Acrylic acid/maleic acid copolymer. (r) Vinylpyrrolidone/vinylimidazole copolymer. Each of the indices x, y, z and w is independently chosen from the integer numbers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, provided that a minimal length of at least two, preferably three building blocks is obtained. While branched backbones such as those depicted in (b) and (c) are not excluded, preference is given to backbones which are not branched. Accordingly, in the formulae for branched backbones such as (b) and (c), preference is given to two indices selected from x, y and z being 0. Potential positions for amino acid side chains are denoted as ellipses in the figure. These positions correspond to the Cαatmons in a peptide.

More generally speaking, the peptidic group may be replaced by a corresponding peptidomimetic. The term “corresponding” in this context means that the peptidomimetic has similar size when compared to a peptidic group. Accordingly, a peptidic group consisting of two amino acids and a peptidic group consisting of three amino acids, respectively, each impose size limits on a corresponding peptidomimetic. A peptidomimetic in accordance with the present invention may use the above mentioned alternative backbones and/or any of the mentioned non-proteinogenic amino acids, noting that amino group and/or carboxylic group of any non-proteinogenic amino acid may be further modified in order to account for the mentioned alternative backbone.

In terms of secondary structure, preference is given to peptidic groups, corresponding peptidomimetics or backbones which are capable of assuming a β-sheet structure. Specific backbones capable of assuming a β-sheet structure include peptide bonds and the alternatives described above.

As disclosed above, X may take the place of the carbonyl group of the α carbon of the C-terminal amino acid of said peptidic group. In other words, and assuming that X is CO which is preferred, X is the carbonyl group of said C-terminal amino acid. In the alternative, the α-carbon may be replaced with X. In that case, the C-terminal amino acid of said peptidic group may be viewed as a truncated amino acid. Having said that, it is formally considered as a separate building block and is also referred to as S1 site of the proteasome inhibitor.

The term “peptidic” refers to peptide bonds connecting the amino acids of said group. The C-terminal end of the peptidic group is the end which is connected to X. To the extent the peptide bonds are inverted, i.e. NHCO instead of CONH, this is a preferred embodiment falling under the term “peptidomimetic”.

Detailed exemplary synthesis procedures for two preferred compounds are given in the examples. In the following, general synthesis procedures are disclosed.

Synthesis of active ester: A) Beta-Keto acid formation from Boc-L-amino acid, esterification for the protection of beta-carboxyl moiety. B) Methylation reaction. C) Boc-Deprotection. D) Peptide coupling. E) Saponification of ester to Beta-Keto acid (partially instable). F) Preparation of an active ester with N-Hydroxysuccinimide.

Synthesis of a Beta-Ketoaldehyde: G) Preparation of a Weinreb-Amide from Boc-L-amino acid. H) Gringard reaction to obtain alpha-Dioxacyclopentyl Ketone. Methylation Reaction. I) Boc-Deprotection. J) Peptide coupling. K) Oxidation to Beta-Ketoaldehyde.

Tripeptide Synthesis: L) Protection of the carboxyl group of Boc-L-Amino Acid by esterification. M) Boc-Deprotection. N) Peptide coupling with Boc-L-amino acid. O) Boc-Deprotection. P) Coupling with N-cap acid. Q) Saponification of ester to acid.

As an alternative to R1being a peptidic group or a peptidomimetic, a group binding between subunits β1 and β2 of the proteasome may be used. Preferably, said group is an aryl sulfonamide, aryl preferably being phenyl, aryl being connected to R12as defined in the following. Between said aryl sulfonamide and R12further moieties may be present, said further moieties preferably being isosteric with the corresponding moieties of the particularly preferred group binding between subunits β1 and β2 as disclosed in the following. Particularly preferred is that said group binding between subunits β1 and β2 is 4-[2-(4-R12-oxy-phenyl) quinolin-4-yl carboxamido] benzene N-acetyl sulfonamide. R12is a linker connecting said group binding between subunits β1 and β2 to X, preferably being C5to C10alkyl, C5to C10alkenyl or —[O—(CH2)2]1-5, wherein one or more C atoms in said linker may be replaced with O and/or N.

Said alkenyl group may comprise one, two or more double bonds.

So far, the group Y═Z has been displayed as a double bond, wherein one of the two bonds comprised in the double bond is dashed. This indicates that Y and Z may either be connected by a double bond or a single bond.

In a preferred embodiment, Y═Z is Y═Z, and preferably selected from CH═O or CH═CH2. In an alternative, Y═Z is Y—Z and is preferably selected from CH2—I, CH2—Br, CH2—Cl, CH2—OPO(OH)2, CH2—OTs or CO—NHS wherein OTs is p-toluene sulfonyloxy and NHS is N-oxy-succinimide. As an alternative to the NHS-activated ester, also other activated esters known in the art may be used.

In a further alternative preferred embodiment, Y═Z is Y—Z and preferably selected from O—I, O—Br, O—Cl, S—I, S—Br and S—I.

In a further preferred embodiment, R2and R3are identical and preferably methyl, H, methoxy or —CH2OH. Particularly preferred is that both R2and R3are methyl or that both R2and R3are H. Especially preferred is that both R2and R3are methyl.

Preferred is that X is CO.

Preferred is that Y═Z is CH═O. Also preferred is that Y═Z is CO—NHS.

Particularly preferred is that X is C═O and Y—Z is CH═O or CO—NHS.

Particularly preferred is that X is C═O and Y—Z is CH═O or CO—NHS and both R2and R3are methyl.

The following preferred embodiments are dedicated to preferred implementations of the targeting moiety R1. Any of the preferred embodiments of R1can be combined with any of the preferred embodiments of X, Y, Z, R2and R3.

In a preferred embodiment, said peptidic group consists of three α-amino acids, wherein preferably (a) the N-terminal amino acid is selected from Ser(OMe), Leu, Phe and Ala; the middle amino acid is selected from Ser(OMe), Leu, Phe and Ala; and/or the C-terminal amino acid is selected from Phe, Tyr, Leu, Ser(OMe) and Ala; or (b) said peptidic group consists of Ser(OMe)-Ser(OMe)-Phe, Leu-Leu-Tyr or Ala-Ala-Ala.

Said N-terminal amino acid is also referred to as the moiety defining the S3 site of the inhibitor. Said middle amino acid defines the S2 site. Said C-terminal amino acid defines the S1 site.

In a further preferred embodiment, said group enhancing delivery is present and is R11—CO, R11—CS—, or R11—SO2—,

R11being selected from the following substituted or unsubstituted groups, groups being carbocyclyl, heterocarbocyclyl, carbocyclyl alkyl, heterocarbocyclyl alkyl, alkyl heterocarbocyclyl alkoxy heterocarbocyclyl, alkoxyalkyl heterocarbocyclyl, heterocarbocyclyl amino, heterocarbocyclyl alkyl heterocarbocyclyl, and alkyl heterocarbocyclyl alkyl heterocarbocyclyl,
wherein alkyl is C1to C4alkyl, preferably methyl, substituents are C1to C4alkyl, preferably methyl or ethyl, C1to C4alkoxy, preferably methoxy or ethoxy, hydroxy and/or halogen, preferably Cl, Br or I, and heteroatoms are O, N and/or S.

R11—CO is particularly preferred.

R11being heterocarbocyclyl is particularly preferred.

The term “carbocyclyl” designates a ring molecule, wherein the ring comprises carbon atoms.

The ring may be formed exclusively by carbon atoms, but does not have to. Accordingly, one, two or more heteroatoms may be present. The ring may be saturated in which case it would be a cyclic alkane, optionally comprising one or more heteroatoms. The ring may contain one, two or more double bonds. If said double bonds are conjugated, carbocyclyl is an aryl moiety.

In a particularly preferred embodiment, (a) said carbocyclyl is aryl or biaryl, aryl being monocyclic or bicyclic and preferably phenyl or naphthyl; and (b) said heterocarbocyclyl is heteroaryl, heteroaryl being monocyclic or bicyclic, bi-heteroaryl, aryl heteroaryl, heteroaryl aryl or heterocycloalkyl, heteroaryl preferably being furyl, thienyl, oxazolyl, isoxazolyl, pyrazolyl, imidazolyl, thiazolyl, pyridinyl, pyrazinyl, pyridazinyl or quinolinyl, aryl preferably being phenyl, heterocycloalkyl preferably being morpholinyl or tetrahydrofuranyl.

Particularly preferred is that heterocarbocyclyl is heteroaryl, in particular monocyclic heteroaryl.

In a particularly preferred embodiment R11is selected from 2-methyl thiazol-5-yl; 4-morpholinyl methyl; 1,4-dichloro 2-phenyl; 6-phenylpyridin-2-yl; pyrazin-2-yl; 3-furyl; 2-thienyl; 5-oxazolyl; 5-isoxazolyl; (5-Me)-3-isoxazolyl; (5-iPr)-3-isoxazolyl; (5-MeOCH2)-3-isoxazolyl; 3-pyrazolyl; 2-imidazolyl; (N-Me)-3-pyrazolyl; (N-Me)-2-imidazolyl; (5-Me)-3-pyrazolyl; 4-pyridinyl; 4-pyridazinyl; 2-(R)-tetrahydrofuranyl; 2-(S)-tetrahydrofuranyl; (5-Me)-3-isoxazolyl-NH—; pyrazin-2-yl; naphth-2-yl; quinolin-2-yl; 4-biphenyl; 3-biphenyl; 4-phenylpyridin-2-yl; 3-phenyl-pyridin-2-yl; 5-phenylpyrazin-2-yl; 6-phenylpyrazin-2-yl; 2-phenyl-thiazol-4-yl; and 5-R111-isoxazol-3-yl;

wherein R111is selected from methyl, 4-morpholinyl methyl, 1,2,4-triazolyl methyl, imidazolyl methyl and N-methyl piperazinyl methyl.

Especially preferred is that R11is 2-methyl thiazol-5-yl.

Especially preferred compounds of the first aspect are the compounds of formulae (IIa) and (IIb):


wherein R1is R11—CO-Ser(OMe)-Ser(OMe)-NH—CH(CH2—C6H5), R11being as defined above, and wherein most preferably R1is 2-methyl thiazol-5-yl carbonyl Ser(OMe)-Ser(OMe)-NH—CH(CH2-C6H5).

In a second aspect, the present invention provides the use of a compound as defined in the first aspect as a proteasome inhibitor.

One or a plurality of compounds in accordance with the first aspect may be used.

Preferably, said proteasome is a proteasome core particle (CP) which is also known as 20S proteasome. In a preferred embodiment, the constitutive core particle (cCP) is used. Alternatively, tissue-specific proteasomal subtypes such as immunoproteasome (iCP) or the thymusproteasome (tCP) may be used.

A proteasome inhibitor in accordance with the present invention is a compound which inhibits one or more proteolytic activities of a proteasome. A proteasome comprises a plurality of proteolytic sites. Preferably, said proteolytic sites are as follows: a site with caspase-like proteolytic activity, a site with trypsin-like proteolytic activity and a site with chymotrypsin-like proteolytic activity. Proteasome inhibitors in accordance with the present invention are capable of inhibiting at least one of these sites. Preferred are compounds which are capable of inhibiting two or all three of these sites.

Related thereto, the present invention provides, in a third aspect, a method of inhibiting a proteasome, said method comprising bringing into contact a proteasome and a compound as defined in the first aspect, provided that methods for treatment of the human or animal body by therapy and diagnostic methods practised on the human or animal body are excluded and/or said method is performed in vitro or ex vivo.

Also provided is a method of inhibiting a proteasome, said method comprising bringing into contact a proteasome and a compound as defined in the first aspect. The method may be performed in vivo.

It is understood that said bringing into contact is effected under conditions which allow a physical contact between said compound and the proteasome. Suitable conditions include aqueous solutions, such as buffered aqueous solutions. Exemplary conditions can be found in the examples enclosed herewith.

The examples include also a proteasome activity assays in accordance with the invention.

A preferred proteasome activity assay is as follows. The activity assay is based on the cleavage of 7-amino-4-methyl-coumarin (AMC) substrates consisting of the fluorophore AMC fused N-terminal to a peptide backbone. These substrates are fluorogenic as the cleaved AMC exhibits fluorescence (emission wavelength: 380 nm; emission wavelength: 460 nm). For every active site a specific substrate is used (LLVY-AMC (SEQ ID NO: 1) for the chymotryptic-like site, LLE-AMC for the caspase-like site, and RLR-AMC for the tryptic-like site). Proteasome activity is monitored by first mixing inhibitor and substrate simultaneously in assay buffer and incubation for 3 min at 37° C. After the addition of 50 nM proteasome and mixing, the increase in fluorescence (excitation wavelength: 380 nm; emission wavelength: 460 nm) is monitored with a spectrofluorimeter. DMSO concentration is kept≤2% for all measurements. The fluorescence readout correlates with proteasome activity. This procedure is utilized to determine the first-order rate constant of inhibition. The IC50, which refers to the inhibitor concentration needed to decrease enzyme activity by 50%, can be determined by measuring proteasome activity at varying inhibitor concentrations.

In a fourth aspect, the present invention provides a medicament or lead compound for developing a medicament comprising or consisting of a compound as defined in the first aspect.

The terms “medicament” and “pharmaceutical composition” are used equivalently herein. A pharmaceutical composition in accordance with the present invention may comprise a pharmaceutically acceptable carrier, diluent or excipient.

Examples of suitable pharmaceutically acceptable carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. It is particularly preferred that said administration is carried out by injection. The compositions may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Pharmaceutically active matter may be present in amounts between 1 ng and 10 mg/kg body weight per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute. Preferred doses are in the range from about 10 to about 40 mg/m2body surface, which correspond to about 0.25 to about 1 mg/kg body weight.

One or more compounds in accordance with the first aspect of the present invention may be the only pharmaceutically active agent(s) comprised in a medicament in accordance with the present invention. Alternatively, one, two or more further pharmaceutically active agents may be present. Said further pharmaceutically active agents are preferably selected from known proteasome inhibitors and/or therapeutic agents for the treatment of multiple myeloma. Known proteasome inhibitors are discussed above and include bortezomib, carfilzomib, ixazomib, marizomib, oprozomib, delanzomib, epoxomicin, dihydroeponemycin. Exemplary agents for the treatment of multiple myeloma are lenalidomide and dexamethasone as well as the combination of the latter two.

The development of a lead compound into a medicament is also known as lead optimization.

Methods for the optimization of the pharmacological properties of lead compounds are known in the art and comprise a method of modifying a lead compound to achieve: (i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmacokinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by (i) esterification of carboxyl groups, or (ii) esterification of hydroxyl groups with carboxylic acids, or (iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi-succinates, or (iv) formation of pharmaceutically acceptable salts, or (v) formation of pharmaceutically acceptable complexes, or (vi) synthesis of pharmacologically active polymers, or (vii) introduction of hydrophilic moieties, or (viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or (ix) modification by introduction of isosteric or bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi) introduction of branched side chains, or (xii) conversion of alkyl substituents to cyclic analogues, or (xiii) derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi) transformation of ketones or aldehydes to Schiff's bases, oximes, acetales, ketales, enolesters, oxazolidines, thiazolidines or combinations thereof.

The various steps recited above are generally known in the art. They include or rely on quantitative structure-action relationship (QSAR) analyses (Kubinyi, “Hausch-Analysis and Related Approaches”, VCH Verlag, Weinheim, 1992), combinatorial biochemistry, classical chemistry and others (see, for example, Holzgrabe and Bechtold, Deutsche Apotheker Zeitung 140(8), 813-823, 2000).

In a fifth aspect, the present invention provides a compound as defined in the first aspect for use in a method of treating, ameliorating or preventing cancer, an autoimmune disease, muscular dystrophy, emphysema, or cachexia accompanying cancer or AIDS.

More generally speaking, the compounds in accordance with the invention are useful in methods of treating, ameliorating or preventing any condition which is amenable to treatment, prevention or amelioration via inhibition of a proteasome.

In a preferred embodiment, said cancer is a lymphoid malignancy, preferably selected from multiple myeloma (MM) including relapsed and refractory MM; non-Hodgkin lymphoma such as B-cell lymphomas including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL), and Waldenström macroglobulinaemia.

In a further preferred embodiment said autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, Sjörgen's syndrome or scleroderma.

In a sixth aspect, the present invention provides a method of identifying a compound capable of inhibiting a proteasome, said method comprising bringing into contact a test compound of formula (III)


wherein R4is an organic group, and X, Y, Z, R2and R3are as defined in accordance with the first aspect or preferred embodiments thereof,
with a proteasome, wherein a decreased activity of the proteasome in presence of said test compound as compared to the absence thereof is indicative of said test compound being a compound capable of inhibiting a proteasome.

This aspect relates to a screening method. Screening may be a biochemical screen or a cellular screen. Generally speaking, a variety of assay designs are available for the purpose of identifying compounds which are capable of inhibiting the activity of a given target molecule, here a proteasome. One of the established distinctions is between cellular assays and biochemical assays. Cellular assays are sometimes viewed as mimicking closer the in vivo situation, however, they suffer from the drawback that any candidate compound generally has to pass the cell membrane in a first step. Biochemical assays are simpler in that respect. The target, here a proteasome, may be presented in enriched or purified form in aqueous solution. In such an assay scenario there is no membrane barrier. The conditions, though, may be further remote from the environment in the organism to be subjected to therapy.

A negative control for the screen is the absence of any test compound as disclosed above. As positive controls one or more compounds in accordance with the first aspect or known proteasome inhibitors as disclosed herein may be used.

As known in the art, screening methods may be implemented in a high throughput fashion.

Accordingly, said method may be effect in a high throughput format. High throughput assays generally may be performed in wells of microtiter plates, wherein each plate may contain 96, 384 or 1,536 wells. Handling of the plates, including incubation at temperatures other than ambient temperature, and bringing into contact of these compounds with the assay mixture is preferably effected by one or more computer controlled robotic systems including pipetting devices. In case large libraries of test compounds are to be screened and/or screening is to be effected within short time, mixture of, for example 10, 20, 30, 40, 50 or 100 test compounds may be added to each well. In case a well exhibits biological activity, in the present case inhibition of a proteasome, said mixture of test compounds may be de-convoluted to identify one or more test compounds in said mixture giving rise to said activity.

A compound capable of inhibiting a proteasome is also referred to as proteasome inhibitor.

Inhibition preferably amounts a decrease of activity of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. Further preferred is a 102-fold, 103-fold, 104-fold, 105-fold, 106-fold, 107-fold, 108-fold or 109-fold reduction of activity.

An art-established measure of inhibitory activity is the IC50value which is the inhibitor concentration where 50% inhibition occurs. Preferably, proteasome inhibitors to be identified by the method of identifying of the present invention as well as compounds in accordance with the first aspect exhibit IC50values in the one digit μM range, preferably below 1 μM, more preferably below 100 nM, below 10 nM, below 1 nM, or below 100 μM. IC50values are preferably determined using the proteasome activity assay as described above.

In accordance with the method of the sixth aspect, R4is not particularly limited. In fact, one of the applications of this method is the identification of further targeting moieties which targeting moieties may be different from the targeting moieties defined in relation to the first aspect of the invention.

As noted above, R4is an organic group. It may have a molecular weight between 200 and 1,000 Da.

In a preferred embodiment, said activity is a proteolytic activity, preferably selected from caspase-like, trypsin-like and chymotrypsin-like activity.

In a further preferred embodiment said proteasome is comprised in an in vitro or ex vivo cell.

This preferred embodiment refers to a cellular assay.

A preferred cellular assay is as follows. Cells (e.g. HEK 293) are transfected with the pZsProSensor-1 vector (Clontech Laboratories, Inc.), a reporter gene construct for proteasomal activity. This vector encodes for a destabilized green fluorescent protein variant (ZsGreen) fused C-terminally to a specific degradation motif mediating the recognition and degradation by the proteasome. The fluorescence readout (excitation wavelength: 493 nm; emission wavelength: 505 nm) of expressed ZsGreen is used to monitor proteasome activity. Inhibition of the proteasome causes accumulation of ZsGreen correlating with an increased fluorescence signal in the GFP channel.

In a seventh aspect, the present invention provides a compound of formula (IV)


wherein
A is selected from NH—NH2, N3and a click chemistry functional group;
X, Y, Z, R2and R3are as defined above.

In an eighth aspect, the present invention provides the use of a compound of formula (IV)


wherein
A is selected from NH—NH2, N3and a click chemistry functional group;
X, Y, Z, R2and R3are as defined above;
in the synthesis of a proteasome inhibitor, of a pharmaceutically active agent or of a lead compound for the development of a pharmaceutically active agent, said pharmaceutically active agent preferably being for use in a method of treating or preventing cancer or an autoimmune disease.

The latter two aspects of the invention are directed to the headgroup or warhead of proteasome inhibitors in accordance with the present invention.

Generally speaking, A is a reactive group. Art-established reactive groups may be used. The purpose of A is to provide robust irreversible coupling to targeting moieties such as R1as defined above. The reactive group A may be used for coupling to any targeting moiety. Upon coupling to a targeting moiety, compounds in accordance with the first aspect may be obtained.

“Click-chemistry is an art-established term; see e.g. Kolb et al. (2001) Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40 (11):2004; Sletten et al. (2009) Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality. Angew. Chem. Int. Ed. 48:6998; Jewett et al. (2010) Cu-free click cycloaddition reactions in chemical biology. Chem. Soc. Rev. 39(4):1272; Best et al. (2009) Click Chemistry and Bioorthogonal Reactions: Unprecedented Selectivity in the Labeling of Biological Molecules. Biochemistry. 48:6571; and Lallana et al. (2011) Reliable and Efficient Procedures for the Conjugation of Biomolecules through Huisgen Azide-Alkyne Cycloadditions. Angew. Chem. Int. Ed. 50:8794.

As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim1reciting 3 alternatives A, B and C, a dependent claim2reciting 3 alternatives D, E and F and a claim3depending from claims1and2and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

Similarly, and also in those cases where independent and/or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim1, a dependent claim2referring back to claim1, and a dependent claim3referring back to both claims2and1, it follows that the combination of the subject-matter of claims3and1is clearly and unambiguously disclosed as is the combination of the subject-matter of claims3,2and1. In case a further dependent claim4is present which refers to any one of claims1to3, it follows that the combination of the subject-matter of claims4and1, of claims4,2and1, of claims4,3and1, as well as of claims4,3,2and1is clearly and unambiguously disclosed.

