1,2,3-triazole binding compounds for klhdc2 and methods for their use

Abstract

1,2,3-Triazole compounds that are KLHDC2 binding compounds and methods for their use for inhibiting the activity of KLHDC2.

Description

[0001] 1,2,3-TRIAZOLE BINDING COMPOUNDS FOR KLHDC2 AND METHODS FOR THEIR USE

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U.S. Patent Application No. 63 / 556,227 filed February 21, 2024, expressly incorporated herein by reference in its entirety.

[0004] STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0005] This invention was made with government support under Grant No. HDTRA 1-22-1 - 0012 awarded by the Defense Threat Reduction Agency (DTRA). The government has certain rights in the invention.

[0006] BACKGROUND

[0007] KLHDC2 is a ubiquitin ligase enzyme that promotes the degradation of various proteins by binding and modifying them. In humans, aberrant proteins can be deleterious to cells and are cleared by the ubiquitin-proteasome system. A group of C-end degrons has recently been identified in some of these abnormal polypeptides, which are recognized by specific cullin-RING ubiquitin E3 ligases (CRLs). Recently, three crystal structures of a CRL2 substrate receptor have been reported, KLHDC2, in complex with the diglycinc-cnding C-cnd degrons of two early terminated selenoproteins and the N-terminal proteolytic fragment of USP1. The E3 recognizes the degron peptides in a similarly coiled conformation and cradles their C-terminal diglycine with a deep surface pocket. By hydrogen bonding with multiple backbone carbonyls of the peptides, KLHDC2 further locks in the otherwise degenerate degrons with a compact interface and unexpected high affinities. In a competition assay, a 12-amino acid C-end diglycine degron peptide interacts with KLHDC2 with an affinity in the single digit nanomolar range. A need exists for compounds that bind to KLHDC2 and regulate KLHDC2 activity. The present disclosure seeks to fulfill this need and provides further related advantages.

[0008] SUMMARY

[0009] In one aspect, the disclosure provides KLHDC2 binding compounds.

[0010] In certain embodiments, the KLHDC2 binding compounds described herein have formula (I): or a stereoisomer, a pharmaceutically acceptable salt, or a pharmaceutically acceptable ester thereof.

[0011] In another aspect, the disclosure provides methods for inhibiting the activity of KLHDC2 using the binding compounds described herein. In the methods, the binding compounds inhibit KLHDC2 enzymatic activity (e.g., enzymatic activity) by binding to KLHDC2 thereby inhibiting the ability of KLHDC2 to engage in protein-protein interactions.

[0012] In certain embodiments, the disclosure provides a method for inhibiting KLHDC2 activity in a subject, comprising contacting KLHDC2 with an amount of a compound of formula (I), or a stereoisomer, or a pharmaceutically acceptable salt or ester thereof, effective to inhibit KLHDC2 activity by inhibiting the KLHDC2-protein substrate binding interaction.

[0013] DETAILED DESCRIPTION

[0014] KLHDC2 is a ubiquitin ligase enzyme that promotes the degradation of various proteins by binding and modifying them. The compounds described herein bind to the same site on KLHDC2 where its substrate proteins bind. In doing so, these binding compounds are KLHDC2 ligands that inhibit the interaction between the ubiquitin ligase and its substrate proteins and block the enzymatic activity of KLHDC2.

[0015] In one aspect, the disclosure provides KLHDC2 binding compounds that bind to KLHDC2 with high affinity and inhibit the enzymatic activity of KLHDC2.

[0016] In another aspect, the disclosure provides methods for inhibiting KLHDC2 activity (via inhibiting the KLHDC2-protein substrate binding interaction) using the binding compounds.

[0017] KLHDC2 Binding Compounds

[0018] In one aspect, the disclosure provides KLHDC2 binding compounds (i.e., compounds that bind to KLHDC2 and thereby regulate its activity). These compounds bind to the deep degron-binding pocket of KLHDC2.

[0019] The KLHDC2 binding compounds described herein have formula (I): or a stereoisomer, a pharmaceutically acceptable salt, or a pharmaceutically acceptable ester thereof, wherein

[0020] R1is H or CH3;

[0021] R2is H, C1-C3 alkyl, or C2-C3 alkenyl; and

[0022] R3is -(CH2)n-X-(CH2)m-Y, wherein n is 1-3, m is 0-1,

[0023] X is S or NH(C=O), and

[0024] Y is an aryl or a heteroaryl group, or R3is -phenylene-S-CHo-Z, wherein Z is a heterocyclyl group.

[0025] The term “aryl” refers to monovalent monocyclic or polycyclic aromatic hydrocarbon group. Representative aryl groups include phenyl and naphthyl groups. In certain embodiments, the aryl group is a phenyl group. The term “phenylene” refers to a divalent phenyl group (e.g., 1 ,4-phenylene, 1,3-phenylene).

[0026] The term “heteroaryl” refers to a monovalent monocyclic group having five to six ring atoms having at least one aromatic ring containing one, two, or three ring heteroatoms independently selected from N, O, or S, the remaining ring atoms being C. Representative heteroaryl groups include pyridyl, furanyl, thienyl, thiazolyl, isothiazolyl, triazolyl, imidazolyl, isoxazolyl, pyrrolyl, pyrazolyl, and pyrimidinyl groups. In certain embodiments, the heteroaryl group is a thiophenyl group.

[0027] The term “heterocyclyl” refers to a saturated or unsaturated non-aromatic cyclic group having three to six ring atoms in which one or more ring atoms arc hctcroatoms independently selected from N, O, or S. Representative heterocyclyl groups include tetrahydropyranyl, piperidine, N-methylpiperidin-3-yl, 2-oxo-piperidinyl, piperazine, / V-mcthylpyrrolidin-3-yl. 3-pyrrolidino, morpholino, (hiomorpholino. pyrrolinyl, pyrrolidinyl, and imidazolinyl groups. In certain embodiments, the heterocyclyl group is a tctrahydropyranyl group.

[0028] The aryl, heteroaryl, and heterocyclyl groups may be substituted with one or more substituents. Suitable substituents include C1-C4 alkyl, C1-C4 haloalkyl (e.g., CF3), C1-C3 alkoxy, and halogen (e.g., fluoro, chloro).

[0029] As used herein, “ pharmaceutically acceptable salt” of a compound refers to an ion of the compound (e.g., carboxylate) ionically association with a counterion (e.g., metal ion) acceptable for pharmaceutical administration. A salt of a compound can be formed by the neutralization reaction of an acid and a base. Salts can be derived from a variety of organic and inorganic counter ions well known in the art and include sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate.

[0030] As used herein, “ pharmaceutically acceptable ester” of a compound refers to an ester of the carboxylic acid of formula (I) that is acceptable for pharmaceutical administration.

[0031] In certain embodiments, R1is H.

[0032] In certain embodiments, R2is H, CH3, or CH2CH=CH2.

[0033] In certain embodiments, R1is H and R2is H, R1is H and R2is CH3, R1is CH3and R2is H, or R1is H and R2is CH2CH-CH2.

[0034] In certain embodiments, R3is -(CH2)n-X-(CH2)m-Y, wherein Y is an aryl (e.g., phenyl) group or a heteroaryl (e.g., thiophenyl) group. In certain of these embodiments, the aryl group is a substituted phenyl group. Suitable phenyl substituents include C1-C4 alkyl (e.g., methyl) and halo (e.g., chloro, fluoro) groups. In other of these embodiments, the heteroaryl group is a substituted thiophenyl group. Suitable thiophenyl substituents include C1-C4 alkyl groups and halo (e.g., chloro, fluoro) groups.

[0035] In certain embodiments, R3is -phenylene- S-CH2-Z, wherein Z is a hctcrocyclyl group. In certain of these embodiments, the heterocyclyl group is a tetrahydrofuranyl group.

[0036] In certain embodiments, R3is Representative KLHDC2 binding compounds of formula (I) include those shown in

[0037] Table 1.

[0038] Table 1. Representative KLHDC2 binding compounds of formula (I).

[0039] Compound 1

[0040] Compound 2

[0041] Compound 3

[0042] Compound 4

[0043] Compound 5 Compound 6

[0044] Compound 7

[0045] An assay for evaluating binding to KLHDC2 is described in Example 4. The KLHDC2 binding activities (IC50, pM) of representative KLHDC2 binding compounds of formula (I) are compared in Table 2.

[0046] Table 2. KLHDC2 binding activities (IC50, pM) of representative formula (I) KLHDC2 binding compounds. The preparations of representative KLHDC2 binding compounds described herein are schematically illustrated in Schemes 1 and 2 below.

[0047] Scheme 1 is a schematic illustration of the preparation of representative KLHDC2 binding compounds described herein from a common triazole intermediate.

[0048] Scheme 1. The preparation of representative KLHDC2 binding compounds from a common triazole intermediate.

[0049] Scheme 2 is a schematic illustration of the preparation of representative KLHDC2 binding compounds described herein via click chemistry from acetylenic and azide intermediates.

[0050]

[0051] Scheme 2. The preparation of representative KLHDC2 binding compounds from acetylenic intermediates.

[0052] The preparations of representative KLHDC2 binding compounds described herein are described in Examples 1-3. Example 1 describes General Synthetic Procedures 1 and 2 for the preparation of representative KLHDC2 binding compounds, Example 2 describes the preparation of representative KLHDC2 binding Compound 1, and Example 3 describes the preparation of representative KLHDC2 binding Compound 2.

[0053] K.LHDC2 Activity Inhibition In another aspect, the disclosure provides methods for inhibiting the activity of

[0054] KLHDC2 using the binding compounds described herein. These binding compounds inhibit the interaction between the ubiquitin ligase and its substrate proteins and block the activity of KLHDC2 (via inhibiting the KLHDC2-protein substrate binding interaction).

[0055] In certain embodiments, the invention provides a method for inhibiting KLHDC2 activity in a subject, comprising contacting KLHDC2 with an amount of a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof, effective to inhibit KLHDC2 activity by inhibiting the KLHDC2-protein substrate binding interaction.

[0056] An assay for evaluating binding to KLHDC2 is described in Example 4.

[0057] In another aspect, the disclosure provides a pharmaceutical composition comprising a KLHDC2 binding compound, or a pharmaceutically acceptable salt or ester thereof, as described herein, and a pharmaceutically acceptable carrier (e.g., diluent).

[0058] In further aspects, methods for using the KLHDC2 binding compounds are provided. In one embodiment, the disclosure provides a method for inhibiting the enzymatic activity of KLHDC2, comprising contacting KLHDC2 with an amount of a KLHDC2 binding compound, or a pharmaceutically acceptable salt or ester thereof, as described herein, effective to inhibit KLHDC2 activity.

[0059] The compounds of the disclosure can be administered in any suitable manner. In some embodiments, the compounds can be delivered locally (e.g., topically) or systemically. In some embodiments, the compounds are administered orally. In some embodiments, the compounds are administered topically, intravenously, or subcutaneously. A physiologically or pharmaceutically acceptable carrier or vehicle can be used to formulate the compound for administration and can be selected according to the mode of administration. In some embodiments, the compounds are delivered orally together with a suitable pharmaceutically acceptable carrier (e.g., at a predetermined dose). The following examples are provided for the purpose of illustrating, not limiting the disclosure.

[0060] EXAMPLES

[0061] Example 1

[0062] General Synthetic Procedures

[0063] The following general synthetic procedures describe the preparation of representative KLHDC2 binding compounds.

[0064] General Synthetic Procedure 1

[0065] The following is a general procedure for the preparation of KLHDC2 binding compounds described herein.

[0066] / 0YA'NH2+

[0067] O

[0068] Reagent 1 Reagent 2

[0069] Reagent 1 (1 equiv.), Reagent 2 (1.05 equiv.), EDC (1.2 equiv.), and HOAt (approximately 1.2- 1.5 equiv. as a stock solution in DMSO) were mixed in dry DMSO (approximately 0.5 mL per 100 mg of product). The reaction mixture was sealed and left at 25 °C for 18 hours. The solvent was evaporated under reduced pressure. Then potassium hydroxide (5 equiv.) (as a 10% solution in methanol) was added. The reaction mixture was sealed and stirred at 50 °C for 6 hours. After cooling to ambient temperature trifluoroacetic acid was added dropwise until neutral pH. Then the mixture was evaporated under reduced pressure and the residue was dissolved in DMSO (approximately 1 mL up to 100 mg of product). The solution was filtered, analyzed by LCMS, and transferred for HPLC purification.

[0070] General Synthetic Procedure 2

[0071] The following is a second general procedure for the preparation of KLHDC2 binding compounds described herein.

[0072] / o o

[0073] Reagent 1 Reagent 2 Reagent 3

[0074] Reagent 1 (1 equiv.), Reagent 2 (1.05 equiv.), diisopropylethylamine, and HATU were mixed in dry DMF (approximately 0.7 mL per 100 mg of product). In case of using a salt of any of reagents, an additional amount of DIPEA was added to the reaction mixture to transfer the reagent to the base form. The reaction mixture was sealed and stirred at ambient temperature for 16 hours. The solvent was evaporated under reduced pressure. The residue was extracted with ethyl acetate (approximately 3 mL per 100 mg of product). Organic layer was washed with brine, separated, and evaporated under reduced pressure. Then Reagent 3, CU(OAC)2* 2H2O (0.1 equiv.), ascorbic acid (1 equiv.), diisopropylethylamine (typically 3 equiv.) and DMF (approximately 0.7 mL per 100 mg of product) were added to the residue. The mixture was sealed and stirred at 90 °C for 16 hours. After cooling to ambient temperature, the solvent was evaporated under reduced pressure and the residue was dissolved in DMSO (approximately 1 mL). Then tetramethylammonium hydroxide (typically 4 equiv.) (as a 25% stock solution in water) was added to the mixture. The reaction mixture was sealed and stirred at 40 °C for 6 hours. After cooling to ambient temperature trifluoroacetic acid was added dropwise until neutral pH. Then the mixture was evaporated under reduced pressure and the residue was dissolved in DMSO (approximately 1 mL up to 100 mg of product). The solution was filtered, analyzed by LCMS, and transferred for HPLC purification.

[0075] Example 2

[0076] The Preparation of a Representative KLHDC2 Binding Compound: 2- {4- 1(2-11(4- tert-butylphenyl)methyl] sulfanyl ) acetamidolmethyll- 1H- 1 ,2,3-triazol- 1 -yl ) acetic acid

[0077] In this example, the preparation of a representative KLHDC2 binding compound, 2- { 4- [(2- { [(4-tert-butylphenyl)methyl] sulfanyl } acetamido)methyl] - 1H- 1 ,2,3-triazol- 1- yl} acetic acid (Compound 1), is described.

[0078] 2- { 4- [(2- { [(4-tert-butylphenyl)methyl] sulfanyl } acetamido)methy 1] - 1 H- 1 ,2,3-triazol-

[0079] 1 -yl} acetic acid was obtained by General Synthetic Procedure 1 using 68 mg (0.281 mmol) of methyl 2-[4-(aminomethyl)-lH-l,2,3-triazol-l-yl]acetate dihydrochloride (Reagent 1), 73 mg (0.307 mmol) of 2- { }(4-tert-butylphenyl)methylJ sulfanyl} acetic acid (Reagent 2), 51 mg (0.329 mmol) of EDC, and 112 mg (1.108 mmol) of triethylamine. Purified by HPLC on Chromatorex 18 SMB 100-5T (Waters) using gradient from 25% to 50% of phase B in phase A (A - 0.1 % trifluoroacetic acid in water, B - 0.1 % trifluoroacetic acid in the mixture of MeCN and water (vol% 95:5)). Yield 44.1 mg (42%), LCMS Purity: 100% (Calc. MW: 376.48, found pos.: 377, found neg.: 375.2, 411), NMR Purity: 95%. Example 3

[0080] The Preparation of a Representative KLHDC2 Binding Compound: 2-r4-({2-F(3.5- dimethylthiophen-2-y Dformamidol acetamido 1 methyl)- 1 H- 1 ,2.3-triazol- 1 -yll acetic acid

[0081] In this example, the preparation of a representative KLHDC2 binding compound, 2- [4- ( { 2- [(3 ,5 -dimethy lthiophen-2-yl)formamido] acetamido } methyl)- 1 H- 1 ,2,3- triazol- 1 -y 1] acetic acid (Compound 2), is described.

[0082] 2-[4-({2-[(3,5-dimethylthiophen-2-yl)formamido]acetamido}methyl)-lH-l,2,3- triazol-l-yl] acetic acid was obtained by General Synthetic Procedure 2 using 59 mg (0.399 mmol) of 2-amino-N-(prop-2-yn-l-yl)acetamide hydrochloride (Reagent 1), 69.9 mg (0.448 mmol) of 3,5-dimethylthiophene-2-carboxylic acid (Reagent 2), 49.1 mg (0.427 mmol) of methyl 2-azidoacetate (Reagent 3), 170.4 mg (0.448 mmol) of HATU, 193 mg (1.494 mmol) of diisopropylethylamine, 165.4 mg (1.281 mmol) of diisopropylethylamine, 8.5 mg (0.043 mmol) of CU(OAC)2‘ 2H2O, 75.1 mg (0.427 mmol) of ascorbic acid, and 155.6 mg (1.708 mmol) of tetramethylammonium hydroxide. Yield 48 mg (32%), LCMS Purity: 100% (Calc. MW: 351.38, found pos.: 352.2, found neg.: 350, 366, 393.8).

[0083] Example 4

[0084] Representative KLHDC2 Binding Assay

[0085] The following is a description of an assay for evaluating binding to KLHDC2.

[0086] Amplified Luminescence Proximity Homogenous Assay (AlphaLISA) was used to monitor protein-protein interaction of two tagged components that are immobilized on beads. One component was GST-KLHDC2 (E3) and the other one was biotinylated-SelK peptide with a length of 12 amino acids ([Biotin] HLRGSPPPMAGG(C) (Bio-Synthesis, Inc.). When the two components bind, they bring the Alpha beads in close proximity to each other that results in a luminescent signal. A compound that has affinity for KLHDC2 will compete with the biotinylated-SelK peptide, preventing the beads from being in proximity to each other, therefore, reducing the luminescent signal in a dose dependent manner. The effects of DMSO on the AlphaLISA readout were tested and it was established that this organic solvent used to dissolve most of the hit compounds has detectable but marginal effects on the assay. When DMSO was kept below 5%, its effect on the AlphaLISA assay was negligible.

[0087] AlphaLISA assays for determining and measuring protein-protein interactions were performed using EnSpire reader (PerkinElmer). GST-tagged KLHDC2 was attached to anti-GST AlphaLISA acceptor beads. Synthetic biotinylated 12 aa SelK degron peptide (Bio-Synthesis, Inc.) was immobilized to streptavidin-coated AlphaLISA donor beads. The donor and acceptor beads were brought into proximity by the interactions between the SelK peptide and KLHDC2. Excitation of the donor beads by a laser beam of 680 nm promotes the formation of singlet oxygen. When an acceptor bead is in close proximity, the singlet oxygen reacts with thioxcnc derivatives in the acceptor beads and causes the emission of 520-620 nm photons, which are detected as the binding signal. If the beads are not in close proximity to each other, the oxygen will return to its ground state and the acceptor beads will not emit light. Competition assays were performed in the presence of numerous compounds, which were titrated at various concentrations.

[0088] The experiments were conducted with 3.83 nM of GST-KLHDC2 and 5.55 nM biotinylated 12 aa SelK peptide in the presence of 5 pg / ml donor and acceptor beads in a buffer of 25 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM TCEP, 0.1% Tween-20, and 0.05 mg / ml Bovine Serum Albumin. The compound concentrations used in competition assays ranged from 15 11M to 1.5 mM. The experiments were done in triplicates. IC50 values were determined using non-linear curve fitting of the dose-response curves generated with Prism 8 (GraphPad).

[0089] Results for representative KLHDC2 binding compounds of formula (I) are summarized in Table 2.

[0090] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the disclosure.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A compound having formula (I)or a stereoisomer, a pharmaceutically acceptable salt, or a pharmaceutically acceptable ester thereof, whereinR1is H or CH3;R2is H, C1-C3 alkyl, or C2-C3 alkenyl; andR3is -(CH2)n-X-(CH2)m-Y, wherein n is 1-3, m is 0-1,X is S or NH(C=O), andY is an aryl group or a heteroaryl group, orR3is -phenylene-S-CHi-Z, wherein Z is a heterocyclyl group.

2. The compound of Claim 1, wherein R1is H.

3. The compound of Claim 1, wherein R2is H, CH3, or CH2CH=CH2.

4. The compound of Claim 1, wherein R1is H and R2is H.

5. The compound of Claim 1, wherein R1is H and R2is CH3.

6. The compound of Claim 1, wherein R1is CH3and R2is H.

7. The compound of Claim 1, wherein R1is H and R2is CH2CH=CH2.

8. The compound of any one of Claims 1-7, wherein Y is a phenyl group.

9. The compound of any one of Claims 1-7, wherein Y is a thiophenyl group.

10. The compound of any one of Claims 1-7, wherein Z is a tetrahydrofuranyl group.

11. The compound of any one of Claims 1-7, wherein R3is12. A compound of Claim 1 selected from the group consisting of:

13. A pharmaceutical composition comprising a compound of any one ofClaims 1-12, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier.

14. A method for inhibiting the activity of KLHDC2, comprising contacting KLHDC2 with an amount of a compound of any one of Claims 1-12, or a pharmaceutically acceptable salt or ester thereof, effective to inhibit KLHDC2 activity.

Images