A policyclic compound and its use

Abstract

The subject of the invention is a compound with general formula (I). The invention also comprises the use of the compound having general formula (I) in medical diagnostics. Another subject of the invention is the use of said compound as a molecular probe for deoxyhypusine synthase interactions.

Description

[0001] A policyclic compound and its use

[0002] The invention relates to pyrochilone derivatives constituting ligands specifically binding to the interface of the enzyme deoxyhypusine synthase (DHS). Due to the high specificity of the resulting complex, the invention may be used in a number of scientific and clinical studies related directly or indirectly to the (DHS) cellular localization and its interaction with other proteins. As has been demonstrated experimentally, the compounds of the invention act by direct and specific binding to DHS. Significantly, it has also been shown that this binding does not lead to the inhibition of catalytic activity of the enzyme.

[0003] Deoxyhypusine synthase (DHS) is a crucial enzyme responsible for the first step of biosynthesis of hypusine - a unique amino acid that is formed as a result of post -translational modification of the eukaryotic translation initiation factor 5A (el F5 A). This process comprises the transfer of aminobutyl moiety from spermidine to a specific lysine residue in elF5A (K50 in the human protein), which leads to the formation of deoxyhypusine. Next, the enzyme deoxyhypusine hydroxylase (DOHH) catalyzes the hydroxylation of this product into fully functional hypusine, which is necessary for the biological activity of elF5A.

[0004] elF5A plays a crucial role in the regulation of translation by controlling the elongation and termination of protein synthesis, especially those containing proline-rich motifs. The proteins regulated by elF5A are involved in fundamental biological processes, such as cell proliferation, cellular stress, and apoptosis. Therefore, dysregulation of the hypusination pathway leads to serious biological and clinical consequences.

[0005] The enzyme DHS is a potential therapeutic target in human diseases. In neoplastic diseases, DHS activity is significantly increased in many types of neoplasms, suggesting that elF5A hypusination may promote neoplastic proliferation and resistance to metabolic stress. Inhibition of DHS may limit the translation of pro-oncogenic proteins, slowing the growth of neoplastic cells and potentially increasing their susceptibility to antineoplastic therapies. For example, studies have shown that pharmacological inhibition of DHS can inhibit the growth of colorectal neoplasms, gliomas, and certain hematological neoplasms. In the case ofneurodegenerative diseases, mutations in the DHPS gene, which encodes DHS, lead to severe neurodegenerative and neurodevelopmental disorders. Deficiency in elF5A hypusination affects the translation of proteins critical for neuronal functions, leading to symptoms such as intellectual developmental delay, epilepsy, and movement disorders. Furthermore, polyaminopathies resulting from abnormal polyamine metabolism are linked to the degeneration of neurons and impaired mitochondrial function. Consequently, selective inhibition of DHS may be a crucial therapeutic strategy in the treatment of neurodegenerative diseases such as Parkinson's disease or Alzheimer's disease. Conversely, in metabolic diseases, elF5A hypusination regulates the expression of proteins involved in lipid synthesis, glycolysis, and amino acid catabolism. Disregulation of DHS leads to metabolic disorders, such as insulin resistance, type 2 diabetes, and mitochondrial diseases. Inhibition of DHS can modulate these pathways, improving cellular function in pathological states.

[0006] In addition to their therapeutic application, non-covalent DHS inhibitors can be used as chemical probes for studying the localization of DHS in cells and mapping the hypusination pathway. Due to their strong, but reversible, binding to the enzyme, they can be used in fluorescence imaging techniques for identifying the subcellular distribution of DHS and the dynamics of its activity in response to various stimuli.

[0007] Non-covalent DHS ligands can also function as "warheads" in novel approaches to proximity-based pharmaceuticals, such as PROTACs (Proteolysis Targeting Chimeras). In such a system, a covalent DHS inhibitor could be conjugated to a ligand that recruits enzymes of the ubiquitin-proteasome system (E3 ligase), which would lead to selective degradation of DHS. This approach would allow for the complete elimination of the enzyme from the cell, which could be of significance in antineoplastic or neuroprotective therapies.

[0008] It is also possible to use them for stabilizing transient DHS complexes with other proteins of a regulatory nature. An example of such an interaction is the DHS complex with ERK1 / 2 kinase described in the scientific literature1.Additionally, non-covalent DHS inhibitors can be used in Click Chemistry technology, where specific chemical moieties can enable further modification of the inhibitors under biological conditions, e.g., by conjugating them with fluorescent markers or nanoparticles.

[0009] The technical problem to be solved by the invention is providing a compound that would bind specifically to deoxyhypusine synthase without simultaneously reducing its activity, wherein the binding would not be of a covalent nature.

[0010] The subject of the invention is a compound with general formula (I)

[0011]

[0012] where:

[0013] n is a natural number between 0 and 3;

[0014] X is selected from the group comprising: -C-, -C=, -C≡, -NH- or -O-;

[0015] Y is selected from the group comprising: -C-, -NH- or -O-;

[0016] Z is selected from the group comprising: -C-, -NH- or -O-;

[0017] R1, R5are selected from the group comprising: -H, -alkyl, -aryl, -heteroaryl, -alkenyl, -alkynyl, -COOH; R2, R4are selected from the group comprising: -H, -OH, -O-alkyl, -O-aryl, -O-alkenyl, -O-alkynyl, =0, -NH2, =NH, -NH-alkyl, -N-dialkyl, -NH-alkenyl, -N-dialkenyl, -NH-alkynyl, -N-dialkynyl, or mixed, -CN, COOH, -SO3H;

[0018] R3is selected from the group comprising: -H, =0, -COOH, -OH, -CN, SO3H, -NH2, -alkyl, -aryl, -heteroaryl, -alkenyl, -alkynyl.

[0019] The second subject of the invention is a use of the compound with general formula (I) in medical diagnostics.

[0020] The invention also comprises the use of the compound with general formula (I) as a molecular probe for deoxyhypusine synthase interactions.The currently used DHS protein inhibitor, compound GC7, is an analog of the substrate -spermidine - which in turn is a substrate for at least several other proteins in cells. Accordingly, the subject of the present invention is characterized by its specificity, as it binds specifically to DHS protein but its chemical structure does not resemble that of the natural substrate. A crucial advantage of the compound, confirmed by in vitro studies, is that this compound is not an inhibitor of the hypusination pathway, allowing it to be used as a chemical probe without interfering with the natural biological process.

[0021] The subject of the invention, due to its specificity and selectivity toward DHS, can be used as a probe in biological and diagnostic studies. Its non-covalent nature ensures bond reversibility, which allows dynamic analysis of biological processes without a permanent modification of the enzyme. The high selectivity of these compounds minimizes the risk of nonspecific interactions with other proteins, which translates into precise monitoring of DHS activity under in vitro and in vivo conditions. Due to their appropriate chemical structure, derivatives of the invention can be conjugated with fluorophores, luminescent probes, or other labels, which allows for their use in spectroscopic and fluorescence techniques, as well as in cellular imaging. These compounds find application in high-throughput screening (HTS), where their non-covalent interactions allow for rapid and efficient assessment of DHS activity under various experimental conditions. Contrary to covalent inhibitors, they allow for the temporary modulation of DHS functions without inducing its degradation, which is particularly significant in pharmacological studies and systems biology. In addition, thanks to their high specificity, they can be used to monitor DHS interactions with elF5A and to analyze the influence of regulatory factors, such as the activation of the ERK pathway, on this process.

[0022] Selective non-covalent DHS ligands can also support structural studies, such as crystallography, NMR, or cryo-EM, by stabilizing the enzyme in specific conformations, which facilitates the design of new DHS inhibitors. Their use in structural and functional methods makes them valuable tools in studies of the mechanisms of DHS action and the entire hypusination pathway.All reagents and solvents used in the synthesis came from commercial sources and were used without additional purification (Merck, AmBeed, Angene, ChemPur). The1H and13C NMR spectra were recorded on a Bruker Avance 600 MHz spectrometer. All chemical shifts (6) are shown in ppm, and coupling constants (J) in hertz [Hz], The chemical shifts were referenced to the internal standard TMS or analyzed in relation to solvents such as CDCl₃, MeOD-d₄, and DMSO-d₆. IR spectra were measured using a Thermo Nicolet iS5 FT-IR spectrometer with the ATR technique. The HRMS analysis was conducted on a micrOTOF-QII mass spectrometer (Bruker) using the ESI ionization method. HRMS detection settings: nebulization 0.4 bar, desolvation 180°C, desolvation gas flow rate 4 L / min, capillary potential 3.50 kV. Nitrogen was used for both nebulization and drying gas. The data were obtained in scanning mode in the range from 50 to 3000 m / z. The compounds were purified through column chromatography with the use of a Grace Reveleris X2 flash chromatograph with FlashPure EcoFlex Silica cartridges (50 pm irregular particle size). The course of the reaction was monitored by visual inspection of TLC plates with TLC Merck 60 silica gel at 254 to 365 nm. The HRMS analysis was conducted on a micrOTOF-QII mass spectrometer (Bruker) using the ESI ionization method. HRMS detection settings: nebulization 0.4 bar, desolvation 180°C, desolvation gas flow rate 4 L / min, capillary potential 3.50 kV. Nitrogen was used for both nebulization and drying gas. The data were obtained in scanning mode in the range from 50 to 3000 m / z.

[0023] Embodiments of the invention have been illustrated in the figures, where:

[0024] Fig. la shows a close-up view of the ligand molecule with the corresponding 2Fo-Fc electron density map at the 1σ contour level,

[0025] Fig. lb shows the binding site of compound 6 (DMP7) in the DHS structure (white) at the central location of the tetramer,

[0026] Fig. 2a shows the effect of DMP7 on DHS activity under conditions without prior enzyme preincubation. Recombinant DHS (50 nM) was incubated in a reaction buffer containing NAD and spermidine, and the reaction was initiated by the addition of elF5Al (10 μM). The deoxyhypusination of elF5A was analyzed by the Western blot method using an anti-(deoxy)hypusine antibody. Ponceau S staining was used for loading control and signal normalization. The DHS inhibitor GC-7 (1 mM) was used as a negative control. A quantitativeanalysis of the hypusinated elF5A level from three independent experiments is shown on the right-hand side. In the presence of DMP7, no statistically significant change in DHS catalytic activity was observed when the enzyme had not been preincubated with the compound, Fig. 2b shows the effect of DMP7 on DHS activity after prior enzyme preincubation with the compound. DHS was preincubated with DMP7 for 1 hour or 3 hours prior to the start of the catalytic reaction. After the addition of elF5Al, the reaction was carried out for 9 minutes, followed by detection of the hypusinated elF5A by the Western blot method, as described above. Contrary to conditions without preincubation, the preincubation of DHS with DMP7 led to a significant increase in enzymatic activity compared to the control samples. These results indicate that DMP7 does not directly stimulate the catalysis of DHS, but rather stabilizes the enzyme during incubation, retaining its catalytic activity.

[0027] 1. Example of synthesis of a selected bispirocholine derivative (6)

[0028] All syntheses were carried out according to the procedures summarized in Scheme 1.

[0029]

[0030] Scheme 1. Synthesis pathway for bispirocholine derivative 6 (DMP7). Reagents used and conditions applied: (a) Indoline (1), 3-chloropropionyl chloride, anh. Et3N, anh. DCM, room temperature, overnight, 75%; (b) 2, AlCh, 140°C, 4 hours, 69%; (c) 3, NBS, anh. DMF, 0°C, 2 h, 72%; (d) 3, acetyl chloride, AlCh, anh. DCM, reflux overnight, 74%; (e) 4, 5, BrettPhos Pd G1 complex with MTBE, potassium tert-butanolate, anh. THF, reflux overnight, 61%.1.1. Synthesis of intermediate product 2

[0031] In a round-bottom argonated flask, indoline (25.00 g, 209.8 mmol, 1.0 eq., CAS: 496-15-1, Merck) and anhydrous triethylamine (58.40 mL, 419.6 mmol, 2.0 eq., CAS: 121-44-8, Merck) were placed, followed by the addition of anhydrous dichloromethane (120 mL, CAS: 75-09-, Merck). The obtained mixture was cooled in an ice bath, followed by dropwise addition of 3-chloropropionyl chloride (30.27 mL, 314.7 mmol, 1.5 eq, CAS: 625-36-5, Merck) for one hour. Once the dropwise addition was complete, the content of the flask was heated to room temperature by removing the ice bath, and then left overnight. The next day, the solution was diluted with dichloromethane (500 mL) and transferred to a separatory funnel. The organic layer was washed with 2 M HCI (CAS: 7647-01-0, Merck), 2 M NaOH (CAS: 1310-73-2, Merck), and water, and then dried over anhydrous MgSO4 (CAS: 7487-88-9, ChemPur). After evaporation of the solvent, the product was purified by recrystallization from ethanol (CAS: 64-17-5, Merck), obtaining 2 as a brownish solid with a yield of 75% (33.0 g)

[0032] Rf= 0.47 (SiO2, hexane [CAS: 110-54-3, Merck] / AcOEt [CAS: 141-78-6, Merck], 1 / 1, v / v); m.p.

[0033] = 87-88°C;1H NMR (600 MHz, CDCl3[CAS: 865-49-6, Merck) δ [ppm] 8.22 (d, J = 8.0 Hz, 1H), 7.22 - 7.15 (m), 7.03 (t, J = 7.4 Hz, 1H), 4.04 (t, J = 8.5 Hz, 2H), 3.89 (t, J = 6.9 Hz, 2H), 3.19 (t, J = 8.4 Hz, 2H), 2.88 (t, J = 6.9 Hz, 2H);13C NMR (151 MHz, CDCl3) δ [ppm] 167.8, 142.7, 131.2, 127.7, 124.7, 124.1, 117.1, 48.0, 39.4, 38.8, 28.0; IR (ATR) [cm1]: 3072, 2960, 2918, 1651, 1416, 760.

[0034] 1.2. Synthesis of intermediate product 3

[0035] A round-bottomed flask was washed with argon, after which intermediate product 2 (7.13 g, 34.00 mmol, 1 eq.) and anhydrous AICI3 (24.99 g, 187.49 mmol, 5.5 eq., CAS: 7446-70-0, Merck) were placed in it. The mixture was heated at a temperature of 140°C for 4 hours, then cooled in an ice bath, and a water / ice mixture (100 mL) was added. The aqueous layer was extracted with ethyl acetate (3 x 50 mL), after which the organic layers were combined, dried over anhydrous MgSO4, and the solvent was evaporated. The product was purified by flash column chromatography (SiO2, hexane / AcOEt, gradient), obtaining 3 as a colorless solid with a yield of 69% (4.06 g)Rf = 0.12 (SiO2, hexane / AcOEt, 1 / 1, v / v); m.p. = 104-105°C1H NMR (600 MHz, CDCl3) δ [ppm] 6.94 (d, J = 7.4 Hz, 1H), 6.85 (dd, J = 7.5, 0.5 Hz, 1H), 6.79 (t, J = 7.4 Hz, 1H), 3.94 - 3.87 (m, 2H), 3.08 - 2.98 (m, 2H), 2.81 (t, J = 7.8 Hz, 2H), 2.52 (t, J = 7.8 Hz, 2H);13C NMR (151 MHz, CDCl3) δ [ppm] 167.1, 140.8, 128.5, 124.9, 122.8, 122.8, 119.7, 44.7, 31.2, 27.3, 24.0; IR (ATR) [cm1]: 3046, 2913, 2900, 1644, 1390.

[0036] 1.3. Synthesis of intermediate product 4

[0037] Intermediate product 3 (1.16 g, 6.70 mmol, 1 eq) was placed in a round-bottom flask and dissolved in anhydrous DMF (10 mL, CAS: 68-12-2, Merck). The mixture was cooled in an ice bath, and a solution of NBS (1.25 g, 7.03 mmol, 1.05 eq, CAS: 128-08-5, AmBeed) in anhydrous DMF (10 mL) was added dropwise., after which the obtained solution was stirred for 2 hours while cooling in an ice bath. After this time, the mixture was transferred to a separatory funnel with water (30 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over anhydrous MgSO4. The solvent was evaporated, and the product was purified by flash column chromatography (SiO2, hexane / AcOEt, gradient), obtaining it as a yellowish solid with a yield of 72% (1.22 g)

[0038] Rf= 0.30 (SiO2, AcOEt); m.p. = 106-108°C1H NMR (600 MHz, CDCl3) δ [ppm] 7.16 - 7.14 (m, 1H), 7.09 - 7.07 (m, 1H), 4.06 - 4.01 (m, 2H), 3.13 (t, J = 8.5 Hz, 2H), 2.91 (t, J = 7.8 Hz, 2H), 2.62 (t, J = 7.8 Hz, 2H);13C NMR (151 MHz, CDCl3) δ [ppm] 167.3, 140.5, 130.9, 128.3, 126.4, 121.8, 115.4, 45.3, 31.3, 27.6, 24.2; IR (ATR) [cm1]: 3036, 2954, 2898, 1656, 1379, 859.

[0039] 1.4. Synthesis of intermediate product 5

[0040] A round-bottomed flask was washed with argon, after which intermediate product 3 (1.09 g, 5.77 mmol, 1 eq.) and AICI3 (5.39 g, 40.41 mmol, 7 eq.) were placed in it, followed by the addition of anhydrous DCM (30 mL). In the next step, acetyl chloride (0.62 mL, 8.66 mmol, 1.5 eq., CAS: 75-36-5, Angene) was added dropwise, and the mixture was refluxed overnight. The next day, the content of the flask was cooled to room temperature and water / ice mixture (50 mL) was added, followed by extraction with ethyl acetate (3 x 30 mL). The combined organic layers were dried over anhydrous MgSO4, the solvent was evaporated, and the product waspurified by flash column chromatography (SiO2, hexane / AcOEt, gradient). Intermediate product 5 was obtained as a pink solid with a yield of 74% (0.92 g).

[0041] Rf= 0.27 (SiO2, AcOEt); m.p. = 94-96°C1H NMR (600 MHz, CDCl3) δ [ppm] 7.54 (s, 1H), 7.50 (s, 1H), 3.96 - 3.90 (m, 2H), 3.05 (t, J = 8.5 Hz, 2H), 2.85 (t, J = 7.8 Hz, 2H), 2.52 (t, J = 7.8 Hz, 2H), 2.37 (s, 3H);13C NMR (151 MHz, CDCl3) δ [ppm] 196.4, 167.5, 145.3, 132.7, 128.9, 126.6, 123.8, 119.3, 45.4, 31.0, 26.9, 26.2, 23.8; IR (ATR) [cm1]: 3554, 3387, 3064, 2964, 1640, 1585, 1383, 1188.

[0042] 1.5. Synthesis of final product 6 (DMP7)

[0043] In a round-bottom flask washed with argon, intermediate products 4 (0.386 g, 1.53 mmol, 1.10 eq.) and 5 (0.300 g, 1.39 mmol, 1.00 eq.) were placed, followed by the addition of anhydrous THF (10 mL, CAS: 109-99-9, Merck). The obtained mixture was deoxygenated by flushing it with argon for 10 minutes. After this time, potassium tert-butoxide (0.702 g, 6.26 mmol, 4.50 eq., CAS: 865-47-4, Merck) and the BrettPhos Pd G1 adduct with MTBE (0.062 g, 0.07 mmol, 0.05 eq., CAS: 1148148-01-9, AmBeed) were added, followed by refluxing overnight. The next day, the reaction was quenched by adding water (10 mL), the mixture was transferred to a separatory funnel and extracted with chloroform (3 x 15 mL, CAS: 67-66-3, Merck). The organic layer was dried over anhydrous MgSO4, and the product was purified by flash column chromatography (SiO2, AcOEt / MeOH [CAS: 67-56-1, Merck], gradient), obtaining the final molecule 6 as a beige solid with a yield of 61% (0.328 g).

[0044] Rf = 0.17 (SiO2, EtOAc / MeOH, 9 / 1, v / v); m.p. = 198°C (with decomposition);1H NMR (600 MHz, CDCl3) δ [ppm] 7.76 (s, 1H), 7.72 (s, 1H), 6.95 (s, 1H), 6.87 (s, 1H), 4.15 (s, 2H), 4.13 -4.09 (m, 2H), 4.07 - 4.02 (m, 2H), 3.21 (t, J = 8.5 Hz, 2H), 3.15 (t, J = 8.4 Hz, 2H), 3.01 (t, J = 7.8 Hz, 2H), 2.92 (t, J = 7.8 Hz, 2H), 2.70 (t, J = 7.8 Hz, 2H), 2.64 (t, J = 7.8 Hz, 2H);13C NMR (151 MHz, CDCl3) δ [ppm] 196.7, 167.9, 167.6, 145.9, 140.5, 132.5, 129.9, 129.4, 129.4, 127.3, 126.6, 124.5, 124.5, 120.3, 119.9, 45.8, 45.4, 45.0, 31.6, 31.5, 27.8, 27.4, 24.5, 24.3; HRMS (ESI+): m / z calculated for C24H22N2O3[M+Na]+409.1523; found [M+Na]+409.1521; IR (ATR) [cm1]: 2913, 2851, 1656, 1591, 1380.

[0045] Example 2. In vitro / in vivo biology2.1. Test methods

[0046] 2.1.1. Cytotoxicity

[0047] For all the derivatives obtained, their influence on the process of hypusination was studied in vitro on isolated proteins and extracts from selected cell lines. The assessment of the hypusination level of the elF5A protein will be conducted according to the method described by Benaceur et al. (2025) in PLoS One2. The study will include an in vitro reaction using purified elF5A, DHPS, and DOHH proteins, as well as spermidine as a substrate. The reaction will be conducted under buffering conditions and then analyzed by the Western blot method using specific antibodies against DHPS, DOHH, hypusine, and elF5A. Additionally, in order to assess the influence of the selected compounds on hypusination, experiments were conducted on cell extracts from HEK293 lines and human fibroblasts. For the quantitative determination of hypusinated elF5A, the Hyp' Assay2method was used, which utilises protein adsorption on 96-well plates, immunodetection with an anti-hypusine antibody, and a color reaction to TMB (3,3',5,5'-tetramethylbenzidine; CAS: 54827-17-7) with absorbance measurement at 450 nm.

[0048] 2.1.2. Specificity

[0049] The ligand design is based on prior high-throughput crystallographic studies3. The study of ligand binding to the DHS protein was based on a standard crystallographic experiment in which a DHS protein crystal obtained under the previously obtained conditions was incubated with a 5 mM solution of the DMP7 compound (the crystal was soaked) for 24 hours. Next, the individually picked crystals were frozen in liquid nitrogen, and the diffraction data were collected at the XALOC beamline at the ALBA synchrotron. Data analysis was carried out using standard crystallography software, i.e., XDS, Phaser, AceDrg, and Refmac5, and visualization and reconstruction of the crystal structure were carried out using COOT program; all of which are available in the CCP4 package. The study was conducted for five separate crystals. Based on these analyses, it was shown that the claimed compound binds specifically to the DHS oligomeric interface in a highly symmetrical manner. The DMP7 compound also exhibits the ability to bind to DHS in a cofactor (NAD)-independent manner.

[0050] 2.1.3. Stability modulation

[0051] Testing of activity levels for all obtained derivatives will be conducted on isolated DHS and DOHH proteins according to the same protocol as described in the point on cytotoxicity.Furthermore, the influence of derivatives on the thermal stability of the protein was analyzed using the Thermofluor method. The Thermofluor4method (thermal shift assay, TSA) involves incubating the tested protein with Sypro Orange dye, which fluorescently marks the exposure of hydrophobic protein regions during denaturation. The samples are then gradually heated in the RT-PCR instrument, and the increase in fluorescence is monitored in real time. The analysis of the melting curve allows the determination of the inflection point, i.e., the melting temperature (Tm) which corresponds to the temperature at which half of the protein is denatured. Tm is an indicator of protein stability and may be used to assess the influence of ligands, mutations, or environmental conditions. A comparison of thermostability showed no differences between the molecule derivatives (Fig. 2), which may indicate that the ligand does not change the thermodynamic equilibrium (that is, exactly Tm), but slows down the deactivation kinetics. The protein may "melt" at the same temperature during slow heating, but at operating temperature (e.g., 37°C), the ligand may prevent small structural fluctuations, which lead to a loss of activity over time.

[0052] 2.2. Stabilization of deoxyhypusine synthase by compound 6

[0053] The influence of compound 6 on the catalytic activity of deoxyhypusin synthase (DHS) was evaluated using an in vitro hypusination test.

[0054] The enzymatic reaction was conducted in a 0.2 M glycine / NaOH buffer at pH 9.2, supplemented with 1 mM DTT, 1 mM NAD, and 1 mM spermidine, at room temperature. DHS was used at a final concentration of 50 nM, and the total reaction volume was 25 μl. The DMP7 compound was prepared as a stock solution in DMSO; consequently, the corresponding control sample contained the same final solvent concentration (4% DMSO).

[0055] In order to examine whether the interaction between 6 and DHS affects the catalytic activity of the enzyme, two experimental variants were analyzed.

[0056] In the first experimental variant, without prior preincubation, the DHS was not incubated with the compound immediately prior to the start of the catalytic reaction.

[0057] (Fig. 3A) The reaction was initiated by adding eIF5A1 to the final concentration of 10 μM using a multichannel pipette. After 9 minutes of incubation, the reaction was stopped by adding 5μl of 6× buffer to the samples for SDS-PAGE. The samples were denatured at the temperature of 95°C for 5 minutes, and then 6 μl of each sample was analyzed by the SDS-PAGE method, followed by the Western blot analysis. Deoxyhypusinated elF5A were detected using an anti-(deoxy)hypusine (Hpu98) antibody. Ponceau S staining was used for normalization of the chemiluminescent signal. Under these conditions, no statistically significant change in the catalytic activity of DHS was observed in the presence of compound 6 compared to the control sample.

[0058] In the second experimental variant, DHS was preincubated with compound 6 for 1 hour or 3 hours prior to the start of the catalytic reaction (Fig. 3B). During the preincubation stage, spermidine was not included in the reaction mixture. After the preincubation was complete, spermidine was added, and the reaction was initiated by adding eIF5A1, as described above. Unexpectedly, it was determined that while prolonged incubation of DHS in the absence of the compound led to a clear decrease in enzymatic activity, the samples preincubated with compound 6 retained significantly higher catalytic activity. Quantitative analysis of hypusinated elF5A levels showed a significantly increased level of the resulting product compared to corresponding control samples without the compound.

[0059] These results show that compound 6 does not act as a direct activator of DHS catalysis, but rather stabilizes the enzyme during incubation, preventing the loss of enzymatic activity over time. In this context, the compounds of the invention may act as pharmacological chaperones, stabilizing DHS and retaining its catalytic competence under conditions that would otherwise lead to partial inactivation of the enzyme.

[0060] Accordingly, the compounds of the invention are able to stabilize DHS activity under conditions in which the enzyme would otherwise undergo partial inactivation. Such a stabilization may be advantageous in pathological conditions associated with reduced DHS activity, including disorders caused by mutations in the DHPS gene, such as deoxyhypusine synthase deficiency.

[0061] The above shows that compound 6 stabilizes deoxyhypusine synthase (DHS) during incubation and retains its catalytic activity. Under standard test conditions, without preincubation, thepresence of compound 6 did not significantly affect the catalytic activity of DHS compared to the control samples. However, when DHS was preincubated with compound 6 prior to the start of the reaction, a significantly higher level of eIF5A deoxyhypusination was observed compared to the corresponding control samples.

[0062] These results indicate that compound 6 does not act as a direct catalytic activator of DHS, but rather stabilizes the enzyme and prevents the loss of its enzymatic activity during incubation. This stabilizing effect suggests that the compounds of the invention can act as ligands capable of retaining the catalytic competence of DHS and therefore may be useful as modulators of the hypusination pathway.

[0063] Literature:

[0064] 1. Becker, A. E. et al. ERK1 / 2 interaction with DHPS regulates elF5A deoxyhypusination independently of ERK kinase activity. Cell Reports 43, 114831 (2024).

[0065] 2. Benaceur, O. et al. Development of a reliable, sensitive, and convenient assay for the discovery of new elF5A hypusination inhibitors. PLoS ONE 20, e0308049 (2025).

[0066] 3. Badger, J. Crystallographic fragment screening. Methods in Molecular Biology 841, 161-177 (2012).

[0067] 4. Huynh, K. & Partch, C. L. Analysis of Protein Stability and Ligand Interactions by Thermal Shift Assay. CP Protein Science 79, (2015).

[0068] Abbreviations:

[0069] Et3N - triethylamine

[0070] DCM - dichloromethane

[0071] NBS - N-Bromosuccinimide

[0072] DMF - N, N-Dimethylformamide

[0073] BrettPhos Pd (G1) – chloro[2-(dicyclohexylphosphino)-3,6-dimethoxy-2′,4′, 6′-triisopropyl-1,1′-biphenyl][2-(2-aminoethyl)phenyl]palladium(II)

[0074] MTBE - methyl tert-butyl ether

[0075] THF - tetrahydrofuranAcOEt - ethyl acetate

[0076] TMS - tetramethylsilane

[0077] MeOD-d4– deuterated methanol DMSO-d6– deuterated dimethyl sulfoxide Rf- delay coefficient

[0078] m.p. - melting point

[0079] NMR - nuclear magnetic resonance

[0080] IR - infrared spectroscopy

Claims

Claims1. A compound with general formula (I)R1R2R4where:n is a natural number between 0 and 3;X is selected from the group comprising: -C-, -C=, -C≡, -NH- or -O-;Y is selected from the group comprising: -C-, -NH- or -O-;Z is selected from the group comprising: -C-, -NH- or -O-;R1, R5are selected from the group comprising: -H, -alkyl, -aryl, -heteroaryl, -alkenyl, - alkynyl, -COOH;R2, R4are selected from the group comprising: -H, -OH, -O-alkyl, -O-aryl, -O-alkenyl, -O- alkynyl, =0, -NH2, =NH, -NH-alkyl, -N-dialkyl, -NH-alkenyl, -N-dia Ikenyl, -NH-alkynyl, -N- dialkynyl, or mixed, -CN, COOH, -SO3H;R3is selected from the group comprising: -H, =0, -COOH, -OH, -CN, SO3H, -NH2, -alkyl, - aryl, -heteroaryl, -alkenyl, -alkynyl.

2. A compound with general formula (I) for use in medical diagnostics.

3. A compound with general formula (I) for use as a molecular probe for deoxyhypusine synthase interactions.

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