ONCOLOGIC AND NEURODEGENERATIVE DISEASES

Field of application

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.

State of the art

In the last two decades, the emergence and development of serious diseases has been shown to be closely associated with changes in the structure of DNA, especially with the degree of its methylation at the 5-position on cytosine bases (5-methylcytosine, 5mC). Epigenetic regulation of the level of 5-methylcytosine and the degree of its oxidation or complete demethylation has a fundamental effect on gene expression. Oxidation of 5- methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), subsequently to 5-formylcytosine (5fC), subsequently to 5-carboxylcytosine (5caC) is catalysed by Ten-eleven translocation enzymes (TET methylcytosine dioxygenases or also TET proteins; TET1-3) [S. Ito, A.C. D'Alessio, O.V. Taranova, K. Hong, L.C. Sowers, Y. Zhang: Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466 (2010) 1129- 1133]. Dysregulation of these processes and abnormal changes in DNA methylation lead to a number of serious pathological conditions. Regulation of TET protein function, especially their inhibition, thus has a direct effect on DNA methylation and its transcription and thus on the development of many diseases, especially oncological and neurodegenerative diseases and disorders, e.g. acute myeloid leukaemia, gliomas, colorectal cancer, breast cancer, melanomas, Rett syndrome, Parkinson's and Alzheimer's disease [H. Cedar, Y. Bergman: Linking DNA methylation and histone modification: patterns and paradigms. Nat. Rev. Genet. 10 (2009) 295-304; L. Tan, Y. G. Shi: Tet family proteins and 5-hydroxymethylcytosine in development and disease. Development 139 (2012) 1895-190; M.M. Suzuki, A. Bird: DNA methylation landscapes: provocative insights from epigenomics. Nat. Rev. Genet. 9 (2008) 465-476; K.D. Rasmusen, K. Helin: Role of TET enzymes in DNA methylation, development, and cancer. Gemes Dev. 30 (2016) 733-750; R.B. Lorsbach, J. Moore, S. Mathew, S.C. Raimondi, S.T. Mukatira, J.R. Downing: 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. Szulwach, L. Lin, C. Street, M. Wunderlich, M. Dawlaty, M.B. Neilly, R. Jaenisch, F.C. Yang, J.C. Mulloy, P. Jin, P.P. Liu, J.D. Rowley, M. Xu, C. He, J. Chen: 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. lacobuzio-Donahue, W.G. Nelson: Global 5-hydroxymethylcytosine content is significantly reduced in tissue stem/progenitor cell compartments and in human cancers. Oncotarget 2 (2011) 627-637; T.L. Cheng, J. Chen, H. Wan, B. Tang, W. Tian, L Liao, Z. Qiu: Regulation of mRNA splicing by MeCP2 via epigenetic modifications in the brain. Sci. Rep. 7 (2017) 42790; E.M. Ellison, E.L. Abner, M.A. Lovell: Multiregional analysis of global 5-methylcytosine and 5- hydroxymethylcytosine throughout the progression of Alzheimer's disease. J. Neurochem. 140 (2017) 383-394; J. Zhao, Y. Zhu, J. Yang, L. Li, H. Wu, P.L. De Jager, P. Jin, D.A. Bennett: A genome-wide profiling of brain DNA hydroxymethylation in Alzheimer's disease. Alzheimers Dement. 13 (2017) 674-688].

TET1 protein (ten-eleven translocation methylcytosine dioxygenase 1) is Fe(ll)- and α- ketoglutarate-dependent dioxygenase that catalysed conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Thus, 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 (2018) Art.No.58]. The possibility of influencing the activity of the TET1 protein may thus be a way to develop new therapeutic systems intended for the treatment of the above-mentioned oncological and neurodegenerative diseases. This strategy is currently limited by the small number of known 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. ChemBioChem 19 (2018) 979-985] or cytosine derivatives [G.N.L. Chua, K. L. Wassarman, H. Sun, J. A. Alp, E. I. Jarczyk, N.J. Kuzio, M.J. Bennett, B.G. Malachowsky, M. Kruse, A.J. Kennedy: Cytosine-Based TET Enzyme Inhibitors. ACS Med. Chem. Lett. 10 (2019) 180-185].

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. [M. Jakubek, Z. Kejík, R. Kaplánek, V. Antonyová, R. Hromádka, V. Šandriková, D. Sýkora, P. Martásek, V. Král: Hydrazones as novel epigenetic modulators: correlation between TET 1 protein inhibition activity and their iron(ll) binding ability. Bioorg. Chem. 88 (2019) 102809]. 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, Ş.G. Küç ükgüzel: Biological Activities of Hydrazone Derivatives. Molecules 12 (2007) 1910-1939; R. León, A.G. Garcia, J. Marco-Contelles: 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. Chem. 10 (2003) 983- 995; A. Gaeta, R.C. Hider: The crucial role of metal ions in neurodegeneration: the basis for a promising therapeutic strategy. Brit. J. Pharmacol. 146 (2005) 1041-1059; G. llppal, S. Bala, S. Kamboj, M. Saini: Therapeutic Review Exploring Antimicrobial Potential of Hydrazones as Promising Lead. Pharma Chem. 3 (2011) 250-268; R. Kaplánek, M. Havlík, J. Rak, J. Králová, V. Král: Tröger's base derivatives and cytostatic properties thereof. Patent CZ305488 B6 (2015); R. Kaplánek, M. Jakubek, M. Havlík, J. Rak, T. Bříza, P. Džubák, M. Hajdúch, P. Konečný, J. Štěpánková, J. Králová, V. Král: Caffeine-8-hydrazones as novel cytostatics for the treatment of oncologic diseases. Patent CZ305625 B6 (2015); G. C. Look, L. Schultz, A. M. Polozov, N. Bhagat, J. Wang, D. E. Zembower, W. F. Goure, T. Pray, G. A. 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. Brit J. Haematol. 43 (1979) 443-449; Z. Kejík, R. Kaplánek, M. Havlík, T. Bříza, D. Vavřinová, B. Dolenský, P. Martásek, V. Král: Aluminium(lll) Sensing by Pyridoxal Hydrazone Utilizing the Chelation Enhanced Fluorescence Effect. J. Lumin. 180 (2016) 269-277; R. Kaplánek, M. Havlík, B. Dolenský, J. Rak, P. Džubák, P. Konečný, M. Hajdúch, J. Králová, V. Král: Synthesis and biological activity evaluation of hydrazone derivatives based on a Tröger's base skeleton. Bioorg. Med. Chem. 23 (2015)

1651-1659].

To monitor the cellular localization of active substances, their molecules must be labeled with a fluorescent label; preferably active substances are used which themselves have fluorescent properties [M. S. T. Goncalves: Fluorescent Labeling of Biomolecules with

Organic Probes. Chem. Rev. 109 (2009) 190-212; Z. Guo, S. Park, J. Yoon, I. Shin: Recent progress in the development of near-infrared fluorescent probes for bioimaging applications.

Chem. Soc. Rev.43 (2014) 16-29; T. Bříza, J. Králová, S. Rimpelová, M. Havlík, R. Kaplánek, Z.

Kejík, B. Reddy, K. Záruba, T. Rumi, I. Mikula, P. Martásek, V. Král: Dimethinium

Heteroaromatic Salts as Building Blocks for Dual-Fluorescence Intracellular Probes.

ChemPhotoChem 1 (2017) 442-450]. 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

Tetraarylpyrrolo[3,2-b]pyrroles - From Serendipitous Discovery to Promising Heterocyclic Optoelectronic Materials. Acc. Chem. Res. 50 (2017) 2334-2345; M. Tasior, B. Koszarna, D. C. Young, B. Bernard, D. Jacquemin, D. Gryko, D. T. Gryko: Fe(lll)-Catalyzed synthesis of pyrrolo[3,2-b]pyrroles: formation of new dyes and photophysical studies. Org. Chem. Front. 6

(2019) 2939-2948].

Combination of the bioactive benzohydrazidove group, which is additionally capable of binding metal ions, and fluorescent pyrrolo[3,2-b] pyrrole core leads to hybrid molecules with potential to affect TET1 protein activity. Their localization in the cells can be monitored by fluorescence microscopy. Novel pyrrolo[3,2-b] pyrroles bearing benzohydrazide group and their use as therapeutics for the treatment of oncological and neurodegenerative disorders and diseases are the subject of this patent.

Summary of invention

The subject thereof are pyrrolo[3,2-b] pyrroles with benzohydrazide substitution of general formula I, where 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 ,

NHCH ₃ , NHCOCH ₃ , or

R1-R2 is -CH=CH-CH=CH-, i.e., condensed benzene ring.

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.

Preparation of pyrrolo[3,2-b] pyrroles with benzohydrazide substitution of general formula I and their cytostatic properties, binding abilities, cellular localisation and inhibitory activity towards TET1 protein are documented in examples below without being limited thereto.

Brief description of the Figures

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 μM) 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.

Examples

Example 1. Preparation ooff 4,4'-( 1,4-di-p-tolyl- 1,4-dihydropyrrolo[3,2-b]pyrrole-2,5- diyl)di(benzohydrazide) (2) of general formula I.

Mixture of methyl 4-formylbenzoate (1 g, 6.1 mmol), p-toluidine (0.65 g, 6.1 mmol) and p- toluensulfonic acid (110 mg, 0.6 mmol) in glacial acetic acid (5 mL) were stirred at 90 °C for 30 min. Then, 2,3-butandion (260 μL, 2.9 mmol) was added to the thick yellow suspension and reaction mixture was stirred at 90 °C for 3 h. After cooling down, suspension was filtered, yellow solid was washed with glacial acetic acid and recrystalized from dichlormethan- hexane mixture (1:1, v/v). Yield was 510 mg (15%) of dimethyl 4,4'-( 1,4-di-p-tolyl-1,4- dihydropyrrolo[3,2-b]pyrrole-2,5-diyl)dibenzoate (intermediate 1).

¹ H NMR (CDCI ₃ ): 7.75 (d, J = 7.7 Hz, 4H), 7.15 (d, J = 8.0 Hz, 4H), 7.10 - 7.01 (m, 8H), 6.35 (s, 2H), 3.77 (s, 6H), 2.27 (s, 6H).

Mixture of diester 1 (500 mg, 0.9 mmol) and catalytical amount of 4-(dimethylamino)pyridine (DMAP; 11 mg, 0.09 mmol) in hydrazine hydrate (9 mL; 180 mmol) was stirred at 115 °C for 2 days. After cooling down, suspension was filtered, yellow solid was washed with cold dichloromethane. Crude product was recrystallized from dichloromethane. Yield was 300 mg (60%) of 4,4'-(1,4-di-p-tolyl-1,4-dihydropyrrolo[3,2-b]pyrrole-2,5-di yl)di(benzohydrazide) (compound 2) in the form of yellow solid.

¹ H ¹ H NMR (DMSO-d ₆ ): 9.70 (s, 2H), 7.98-7.51 (m, 4H), 7.49-6.91 (m, 12 H), 6.55 (s, 2H), 4.57 (s, 4H), 2.35 (s, 6H).

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 2. Preparation of 4,4'-(1,4-bis(4-hydroxyphenyl)-1,4-dihydropyrrolo[3,2-b]pyrr ole- 2,5-diyl)di(benzohydrazide) (3) of general formula I.

Compound was prepared according to method mentioned in Example 1, except that in the first step, 4-aminophenol (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. After recrystallization from chloroform-methanol mmiixxttuurree wwaass obtained 4,4'-(1,4-bis(4-hydroxyphenyl)-1,4- dihydropyrrolo[3,2-b]pyrrole-2,5-diyl)di(benzohydrazide) (3) in the overall yield of 9.0% (relative to 2,3-butanedione).

¹ H NMR (DMSO-d ₆ ): 9.77 (s, 2H), 8.92 (bs, 2H), 8.10-6.90 (m, 16 H), 6.54 (s, 2H), 4.57 (s, 4H). Example 3. Preparation of 4,4'-(1,4-bis(3-chlorophenyl)-1,4-dihydropyrrolo[3,2-b]pyrro le-2,5- diyl)di(benzohydrazide) (4) of general formula I.

Compound was prepared according to method mentioned in Example 1, except that in the first step, 3-chloroaniline (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. After recrystallization from chloroform-methanol mmiixxttuurree wwaass obtained 4,4'-(1,4-bis(3-chlorophenyl)-1,4- dihydropyrrolo[3,2-b]pyrrole-2,5-diyl)di(benzohydrazide) (4) in the overall yield of 8.2% (relative to 2,3-butanedione).

¹ H NMR (DMSO-d ₆ ): 9.66 (s, 2H), 8.00-7.50 (m, 4H), 7.40-7.05 (m, 12 H), 6.60 (s, 2H), 4.52 (s, 4H).

Example 4. Preparation of 4,4'-(1,4-bis(3-tolyl)-1,4-dihydropyrrolo[3,2-b]pyrrole-2,5- diyl)di(benzohydrazide) (5) of general formula I.

Compound was prepared according to method mentioned in Example 1, except that in the first step, 3-methylaniline (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. After recrystallization from chloroform-methanol mixture was obtained 4,4'-(1,4-bis(3-tolyl)-1,4-dihydropyrrolo[3,2- b]pyrrole-2,5-diyl)di(benzohydrazide) (5) in the overall yield of 9.1% (relative to 2,3- butanedione).

¹ H NMR (DMSO-d ₆ ): 9.74 (s, 2H), 8.00-7.50 (m, 4H), 7.50-6.88 (m, 12 H), 6.53 (s, 2H), 4.60 (s, 4H), 2.38 (s, 6H).

Example 55. Preparation of 4,4'-(1,4-diphenyl-1,4-dihydropyrrolo[3,2-b]pyrrole-2,5- diyl)di(benzohydrazide) (6) of general formula I.

Compound was prepared according to method mentioned in Example 1, except that in the first step, aniline (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. After recrystallization from chloroform was obtained 4,4'-(1,4-diphenyl-1,4-dihydropyrrolo[3,2-b]pyrrole-2,5-diyl )di(benzohydrazide) (6) in the overall yield of 7.7% (relative to 2,3-butanedione).

¹ H NMR (DMSO-d ₆ ): 9.80 (s, 2H), 8.00-6.80 (m. 18H), 6.50 (s, 2H), 4.61 (s, 4H).

Example 6. Preparation of 4,4'-(1,4-bis(4-methoxyphenyl)-1,4-dihydropyrrolo[3,2-b]pyrr ole- 2,5-diyl)di(benzohydrazide) (7) of general formula I.

Compound was prepared according to method mentioned in Example 1, except that in the first step, 4-methoxyaniline (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. After recrystallization from dichloromethane was obtained 4,4'-(1,4-bis(4-methoxyphenyl)-1,4-dihydropyrrolo[3,2- b]pyrrole-2,5-diyl)di (benzohydrazide) (7) in the overall yield of 9.4% (relative to 2,3- butanedione).

¹ H NMR (DMSO-d ₆ ): 9.70 (s, 2H), 8.05-6.88 (m, 16 H), 6.53 (s, 2H), 4.58 (s, 4H), 3.81 (s, 6H).

Example 7. Preparation of 4,4'-(2,5-bis(4-(hydrazinocarbonyl)phenyl)pyrrolo[3,2-b]pyrr ole- 1,4-diyl)dibenzamide (8) of general formula I.

Compound was prepared according to method mentioned in Example 1, except that in the first step, 4-aminobenzamide (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. After recrystallization from dichloromethane-methanol mixture was obtained 4,4'-(2,5-bis(4-(hydrazinocarbonyl)phenyl) pyrrolo[3,2-b]pyrrole-1,4-diyl)dibenzamide (8) in the overall yield of 6.9% (relative to 2,3- butanedione).

¹ H NMR (DMSO-d ₆ ): 8.10-6.81 (m, 16 H), 6.56 (s, 2H), 4.62 (bs, 4H).

Example 8. Preparation of 4,4'-(1,4-bis(4-bromophenyl)-1,4-dihydropyrrolo[3,2-b]pyrrol e-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. After recrystallization from dichloromethane-methanol mmiixxttuurree wwaass obtained 4,4'-(1,4-bis(4-bromophenyl)-1,4- dihydropyrrolo[3,2-b]pyrrole-2,5-diyl)di(benzohydrazide) (9) in the overall yield of 10.5% (relative to 2,3-butanedione).

¹ H NMR (DMSO-d ₆ ): 9.87 (s, 2H), 8.05-7.52 (m, 4H), 7.47-6.90 (m, 12 H), 6.50 (s, 2H), 4.57 (s,

4H).

Example 9. Preparation of 4,4 ^, -(1,4 -bis(4-nitrophenyl)-1,4-dihydropyrrolo[3,2-b]pyrrole-2,5- diyl)di(benzohydrazide) (10) of general formula I.

Compound was prepared according to method mentioned in Example 1, except that in the first step, 4-nitroaniline (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. After recrystallization from dichloromethane-methanol mmiixxttuurree was obtained 4,4'-(1,4-bis(4-nitrophenyl)-1,4- dihydropyrrolo[3,2-b]pyrrole-2,5-diyl)di(benzohydrazide) (10) in the overall yield of 6.5% (relative to 2,3-butanedione).

¹ H NMR (DMSO-d ₆ ): 9.85 (s, 2H), 8.05-7.52 (m, 4H), 7.47-6.90 (m, 12 H), 6.47 (s, 2H), 4.60 (s, 4H). Example 10. Preparation of 4,4 ^, -(1,4-bis(4-(dimethylamino)phenyl)-1,4-dihydropyrrolo[ 3,2- b]pyrrole-2,5-diyl)di(benzohydrazide) (11) of general formula I.

Compound was prepared according to method mentioned in Example 1, except that in the first step, 4-(dimethylamino)aniline (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. After recrystallization from dichloromethane-methanol mixture was obtained 4,4'-(1,4-bis(4-dimethylaminophenyl)-1,4- dihydropyrrolo[3,2-b]pyrrole-2,5-diyl)di(benzohydrazide) (11) in the overall yield of 7.3% (relative to 2,3-butanedione).

¹ H NMR (DMSO-d ₆ ): 9.80 (s, 2H), 8.05-6.90 (m, 16 H), 6.53 (s, 2H), 4.58 (s, 4H), 2.88 (s, 12H).

Example 11. Binding properties of pyrrolo[3,2-b] pyrroles with benzohydrazide substitution towards Fe ²⁺ ions.

Absorption spectrum ooff 4,4'-(1,4-di-p-tolyl-1,4-dihydropyrrolo[3,2-b]pyrrole-2,5- diyl)di(benzohydrazide) of general formula I (compounds 2) was measured on Cintra 404 GBC spectrometer. The complexation of compound 2 with iron ions (Fe ²⁺ ) was studied by UV-Vis spectroscopy in an aqueous medium (water/DMSO, 99: 1). The binding constant (Ks) was calculated from the changes in the absorbance of compound 2 at the spectral maximum of the iron complex (Fe ²⁺ ) (391 nm) by non-linear regression analysis using Letagrop Spefo 2005 software. The concentration of compound 2 was 10 μM. 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. The binding constant for the 1:1 complex (Fe ²⁺ : compound 2) was Log (K) = 19.4 and for the 1:2 complex was Log (K) = 8.9, respectively. 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 μM) 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).

Example 12. Cytotoxicity of pyrrolo[3,2-b]pyrroles with benzohydrazide substitution.

IC ₅₀ measurement: Cell viability was measured by the 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 μL Dulbecco's Modified Eagle Medium (DMEM) containing streptomycin and 10% fetal bovine serum (FBS) at 37°C and 5% CO ₂ . 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 μL 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 μM (100 nM, 1 μM, 10 μM, 50 μM and 100 μM). Each concentration was measured four times in one test and the whole test was repeated three times. The chelators were diluted in DMSO and DMEM to a final volume of 200 μL After 48 hours of exposure, the chelator solutions were aspirated and 175 μL of MTT solution was added to the cells. Incubation was for 2 hours. After incubation, 125 μL 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 μM. Compound 2 showed a cytotoxic effect both on healthy human HF-P4 fibroblasts (IC ₅₀ = 23.34 μM) and on human malignant melanoma cells A-2058 (IC ₅₀ = 25.59 μM) and human osteosarcoma cells U2-OS (IC ₅₀ = 1.34 μM).

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. After 1 day of incubation, 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 μM. MitoTracker ^® Red FM at 50 nM (MTR) and LysoTracker ^® Green FM at 300 nM (LTG) were used as standards for assessing accurate intracellular localization. After the incubation period, the cells were washed twice with PBS and left in fresh medium without phenol red. For cell visualization, we used a water lens HC PL APO CS2 63x (NA 1.2) and a laser with an excitation wavelength of 405 nm (power 10%) with a fluorescence emission range of 415-470 nm. For MitoTracker ^® Red we used a laser with an excitation wavelength of 579 nm (power 8%) and for LysoTracker® Green we used a laser with an excitation wavelength of 504 nm (power 10%). We also determined the co-localization of the test substance with mitochondria and lysosomes by correlation statistical analysis of the intensity values of red (MitoTracker) and blue (compound 2) or green (LysoTracker) and blue (compound 2) pixels in two-channel images. Using ImageJ software, we determined Pearson's correlation coefficients.

Using real-time fluorescence microscopy of live cells on the human fibroblast line HF-P4, we determined the mitochondrial localization of compound 2 using an organelle-specific fluorescent label (MTR, LTG). Pearson's correlation coefficient for co-localization of compound 2, and MTR showed a very positive linear relationship, thus confirming the localization of compound 2 in mitochondria. The value of Pearson's correlation coefficient was 0.92.

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.

4,4'-(1,4-di-p-tolyl-1,4-dihydropyrrolo[3,2-b]pyrrole-2,5 -diyl)di(benzohydrazide) of general formula I (compound 2) was tested to determine 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 μM.

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.