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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems

Definitions

• the invention relates to pyrrolo[3,2-b]pyrroles with benzohydrazide substitution and their use as therapeutics for the treatment of oncological and neurodegenerative disorders and diseases.
• TET1 a member of a novel protein family, is fused to MLL in acute myeloid leukemia containing the t(10; 11)(q22;q23). Leukemia 17 (2003) 637- 641; A.P. Feinberg, R. Ohlsson, S. Henikoff: The epigenetic progenitor origin of human cancer. Nat. Rev. Genet. 7 (2006) 21-33; J. Wang, G.M. Hong, A.G. Elkahloun, S. Arnovitz, J. Wang, K.
• TET1 plays an essential oncogenic role in MLL-rearranged leukemia. Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 11994-11999; M.C. Haffner, A. Chaux, A.K. Meeker, D.M. Esopi, J. Gerber, L.G. Pellakuru, A. Toubaji, P. Argani, C.
• TET1 protein (ten-eleven translocation methylcytosine dioxygenase 1) is Fe(ll)- and ⁇ - ketoglutarate-dependent dioxygenase that catalysed conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC).
• TET1 protein participates in the regulation and control of gene expression [J. An, A. Rao, M. Ko: TET family dioxygenases and DNA demethylation in stem cells and cancers. Exp. Mol. Medicine 49 (2017) e323; X. Wu, G. Li, R. Xie: Decoding the role of TET family dioxygenases in lineage specification. Epigenetics Chromatin 11 (2016) Art.No.58].
• TET1 protein effectors For example, thioethers of macrocyclic peptides have been described [K. Nishio, R. Belle, T. Katoh, A. Kawamura, T. Sengoku, K. Hanada, N. Ohsawa, M. Shirouzu, S. Yokoyama, H. Suga: Thioether Macrocyclic Peptides Selected against TET1 Compact Catalytic Domain Inhibit TET1 Catalytic Activity.
• TET1 protein One of the types of compounds that can affect the TET1 protein are substances capable of binding iron ions. Recently, it has been found that hydrazine derivatives can affect (inhibit) the TET1 protein and this property is closely related to their ability to bind iron ions.
• hydrazine derivatives can affect (inhibit) the TET1 protein and this property is closely related to their ability to bind iron ions.
• Hydrazine derivatives namely hydrazides and hydrazones are known for their wide range of biological activity, including anticancer, antimicrobial, antimycobacterial, antiviral, fungicide, antimalarial activities. They can also serve as anti-alzheimer's or anti-parkinson's therapeutics [B. Narasimhan, P. Kumar, D. Sharma: Biological activities of hydrazide derivatives in the new millennium. Acta Pharm. Sci. 52 (2010) 169-180; P. Kumar, B. Narasimhan: Hydrazides/ Hydrazones as Antimicrobial and Anticancer Agents in the New Millennium. Mini-Rev. Med. Chem. 13 (2013) 971-987; J.L. Buss, B.T. Greene, J. Turner, F.M.
• Torti, S.V. Torti Iron Chelators in Cancer Chemotherapy Curr. Top. Med. Chem. 4 (2004) 1623-1635; S. Rollas, ⁇ .G. kuç ükgüzel: Biological Activities of Hydrazone Derivatives. Molecules 12 (2007) 1910-1939; R. León, A.G. Garcia, J. Marco-Concetate: Recent Advances in the Multitarget-Directed Ligands Approach for the Treatment of Alzheimer's Disease. Med. Res. Rev. 33 (2013) 139-189; T.F. Tam, R. Leung-Toung, W. Li, Y. Wang, K. Karimian, M. Spinoet: Iron Chelator Research: Past, Present, and Future Curr. Med.
• Krafft Methods of inhibiting the formation of amyloid-beta diffusable ligands using acylhydrazide compounds.
• Patent US2011098309A 1 Hydrazine derivatives show also binding ability towards metal ions [T. Hoy, J. Humphrys, A. Williams, P. Ponka, A. Jacobs: Effective iron chelation following oral administration of an isoniazid-pyridoxal hydrazone.
• Král Aluminium(lll) Sensing by Pyridoxal Hydrazone Utilizing the Chelation Enhanced Fluorescence Effect. J. Lumin. 180 (2016) 269-277; R. Kaplánek, M. Havl ⁇ k, B. Dolensk ⁇ , J. Rak, P. D ⁇ ubák, P. Kone ⁇ n ⁇ , M. Hajd ⁇ ch, J. Králová, V. Král: Synthesis and biological activity evaluation of hydrazone derivatives based on a Tröger's base skeleton. Bioorg. Med. Chem. 23 (2015)
• Heteroaromatic Salts as Building Blocks for Dual-Fluorescence Intracellular Probes.
• Pyrrolo[3,2-b] pyrroles are example of suitable fluorescent core (probe) [M. Krzeszewski, B. Thorsted, J. Brewer, D. I. Gryko: Tetraaryl-, Pentaaryl-, and Hexaaryl-1,4-dihydropyrrolo[3,2-b] pyrroles: Synthesis and Optical Properties. J. Org. Chem. 79 (2014) 3119-3128; M. Krzeszewski, D. Gryko, D. T. Gryko: The
• R 1, R2 are independently H, OH, C 1 to C6 alkyl, C(CH 3 ) 3 , allyl, benzyl, phenyl, F, Cl, Br, I, CH 2 OH, OCH 3 , OCH 2 CH 3 , CF 3 , OCF 3 , CONH 2 , CONHCH 3 , CON(CH 3 ) 2 , NO 2 , N(CH 3 ) 2 , N(CH 2 CH 3 ) 2 ,
• the compounds of general formula I have cytostatic effects and show inhibitory activity towards TET1 protein and thus they can be used for the preparation of therapeutic systems for the treatment of oncological and neurodegenerative disorders and diseases.
• Figure 1 shows UV-Vis spectrum of compound 2 in the presence of Fe(ll) ions.
• Figure 2 shows titration curve for compound 2 (10 ⁇ M) showing the dependence of the absorbance of the complex at the maximum (390 nm) on concentration of Fe(ll) in aqueous medium (water/DMSO, 99:1).
• Figure 3 shows IC 50 of compound 2 for all tested cell lines.
• Figure 4 shows intracellular localization of compound 2 (left), commercial MitoTrackerRed (middle) and merge of compound 2 and MitoTrackerRed (right).
• Figure 5 shows dependence of normalized TET 1 activity (enzymatic activity in the presence of compound 2 / activity of uninhibited enzyme) on the concentration of compound 2.
• the preparation can also be carried out in such a way that the crude intermediate, after filtration and washing with acetic acid, is used directly in the next reaction step, i.e. it is not purified by crystallization. Only the final product (compound 2) was crystallized. The total yield of compound 2 prepared in this way is comparable to the first one process.
• Example 8 Preparation of 4,4'-(1,4-bis(4-bromophenyl)-1,4-dihydropyrrolo[3,2-b]pyrrole-2,5-diyl)di(benzohydrazide) (9) of general formula I.
• Compound was prepared according to method mentioned in Example 1, except that in the first step, 4-bromoaniline (6.1 mmol) was used instead of p-toluidine. Filtered crude intermediate was used to the next step without further purification. Second step was performed according to the procedure described in Example 1.
• Example 11 Binding properties of pyrrolo[3,2-b] pyrroles with benzohydrazide substitution towards Fe 2+ ions.
• the concentration of compound 2 was 10 ⁇ M.
• the concentration of iron ions (Fe 2+ ) ranged from 0 mM to 0.5 mM.
• the range of the UV-Vis spectrum was 300 to 600 nm, with 1 nm data spacing at a scan rate of 300 nm/min.
• the solutions were mixed in a cuvette throughout the titration and after each addition of iron.
• the ability of test compound (2) to chelate iron ions (Fe 2+ ) was demonstrated.
• Figure 1 shows UV-Vis spectrum of compound 2 in the presence of Fe(ll) ions.
• Figure 2 shows titration curve for compound 2 (10 ⁇ M) showing the dependence of the absorbance of the complex at the maximum (390 nm) on concentration of Fe(ll) in aqueous medium (water/DMSO, 99:1).
• IC 50 measurement Cell viability was measured by the MTT [3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] colorimetric assay.
• MTT MTT [3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] colorimetric assay.
• HF-P4 and A-2058 cells were cultured in 96- well plates at a density of 1.0 x 10 5 cells/well and grown for 24 hours in 200 ⁇ L Dulbecco's Modified Eagle Medium (DMEM) containing streptomycin and 10% fetal bovine serum (FBS) at 37°C and 5% CO 2 .
• DMEM Dulbecco's Modified Eagle Medium
• FBS fetal bovine serum
• U2-OS cells were cultured in 96-well plates at a density of 1.0 x 10 5 cells/well and allowed to grow for 24 hours in 200 ⁇ L of McCoy's medium containing streptomycin and 10% fetal bovine serum (FBS) at 37°C and 5% CO 2 . After incubation, compound 2 was added to the cells from each well in a concentration range of 100 nM to 100 ⁇ M (100 nM, 1 ⁇ M, 10 ⁇ M, 50 ⁇ M and 100 ⁇ M). Each concentration was measured four times in one test and the whole test was repeated three times.
• McCoy's medium containing streptomycin and 10% fetal bovine serum (FBS) at 37°C and 5% CO 2 .
• FBS fetal bovine serum
• the chelators were diluted in DMSO and DMEM to a final volume of 200 ⁇ L After 48 hours of exposure, the chelator solutions were aspirated and 175 ⁇ L of MTT solution was added to the cells. Incubation was for 2 hours. After incubation, 125 ⁇ L of DMSO was added to dissolve the dark formazan crystals formed in intact cells, and the absorbance was measured at 570 nm using a Tegan MicroPlate reader. The effect of the tested compound 2 on healthy human HF-P4 fibroblasts, human malignant melanoma cell A-2058 and human osteosarcoma cell U2-OS was evaluated using the MTT assay. The range of chelator concentrations was 0.1-100 ⁇ M.
• Figure 3 shows IC 50 of compound 2 for all tested cell lines.
• Example 13 Intracellular localization of pyrrolo[3,2-b] pyrroles with benzohydrazide substitution.
• Intracellular localization of 4,4'-(1,4-di-p-tolyl-1,4-dihydropyrrolo[3,2-b]pyrrole-2,5- diyl)di(benz-hydrazidu) of general formula I (compound 2) was examined by real-time fluorescence microscopy of live cells using a Leica TCS SP8 WLL SMD-FLIM microscope at 37°C and 5% CO 2 atmosphere.
• HF-P4 cells at a density of 4.0 x 10 4 cells/slide were seeded on 22x22 mm slides to visualize viable cells in complete cell culture medium (DMEM with streptomycin and 10% FBS). The cells were left overnight (24 hours) for adhesion.
• the medium was aspirated, and the cells were incubated for 15 minutes (37°C, 5% CO 2 ) in DMEM containing compound 2 at a concentration of 1 ⁇ M.
• MitoTracker ® Red FM at 50 nM (MTR) and LysoTracker ® Green FM at 300 nM (LTG) were used as standards for assessing accurate intracellular localization.
• the cells were washed twice with PBS and left in fresh medium without phenol red.
• Figure 4 shows intracellular localization of compound 2 (left), commercial MitoTrackerRed (middle) and merge of compound 2 and MitoTrackerRed (right).
• Example 14 Inhibitory activity of pyrrolo[3,2-b]pyrroles with benzohydrazide towards TET1 protein.
• TET hydroxylase activity (TET1 protein) using a fluorimetric kit. Purified TET 1 enzyme was used. The inhibitory activity of compound 2 was measured according to the manufacturer's protocol. TET 1 binds to a methylated substrate and converts it to hydroxymethylated products that can be recognized by a specific antibody. The amount of these products was determined by measuring the fluorescence intensity on a MicroPlate reader at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. In the experiment, we observed an inhibitory effect for TET 1 for compound 2. The IC 50 value for compound 2 was 0.87 ⁇ M.
• Figure 5 shows dependence of normalized TET 1 activity (enzymatic activity in the presence of compound 2 / activity of uninhibited enzyme) on the concentration of compound 2.

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• 2020
  • 2020-09-22 CZ CZ2020-528A patent/CZ309298B6/cs unknown
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  • 2021-09-21 WO PCT/CZ2021/050099 patent/WO2022063352A1/en not_active Ceased

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