WO2022063351A1 - Azulene hydrazide-hydrazones and their use in the treatment of oncologic diseases - Google Patents

Abstract

The subject of the invention is azulene hydrazide-hydrazones bearing 2-hydroxyaryl group of general formula I. These compounds show selective cytostatic effect and therefore they can be used for the preparation of medicaments for the treatment of oncological diseases.

Description

Azulene hydrazide-hydrazones and their use in the treatment of oncologic diseases

Field of application

The invention relates to azulene derivatives with a hydrazide-hydrazone group and their use as selective cytostatics for the treatment of oncological diseases (leukaemias and cancers).

State of the art

Cancer is one of the most common causes of death in developed countries. In recent years, a new strategy combining therapy and diagnosis, so-called theranostics, has been used to achieve a better prognosis. Iron is known to be an essential element for humans and many other organisms. Iron plays a crucial role in many processes, such as oxygen transport, energy processes, detoxification of chemicals, DNA synthesis. On the other hand, the level of free iron must be strictly regulated, because excess free iron can contribute to oxidative stress due to the formation of free radicals (Fenton's reaction) and can thus lead to tissue damage. Iron-related processes in cancer development and tumour cell growth include cellular metabolism, DNA replication, cell cycle regulation, apoptosis, ferroptosis. As a result, iron is an important and promising target for therapeutic and diagnostic interventions. One way to regulate iron levels is to use chelators.

Chelators are compounds that can strongly bind metal ions, such as iron. These substances are able to form complexes with intra- and extracellular iron. Oxygen and nitrogen donor centres are involved in metal bonding. Several approved chelators are used in human medicine. One of the commonly used is desferrioxamine (DFO), used to treat disorders and diseases associated with chronic iron excess, such as thalassemia. Iron chelation can be an effective approach to reduce the formation of highly toxic hydroxyl radicals, leading to oxidative stress. Chelators can also affect the level of iron necessary for cell growth and division and can thus serve as therapeutics for the treatment of oncological diseases. [T. F. Tam, R. Leung-Toung, W. Li, Y. Wang, K. Karimian, M. Spinoet: Iron Chelator Research: Past, Present, and Future Curr. Med. Chem. 2003, 10, 983-995; D. R. Richardson: Iron chelators as therapeutic agents for the treatment of cancer Crit. Rev. Oncol. Hematol. 2002, 42, 267-81; J. L. Buss, B. T. Greene, J. Turner, F. M. Torti, S. V. Torti: Iron Chelators in Cancer Chemotherapy Curr. Top. Med. Chem. 2004, 4, 1623-1635; D. S. Kalinowski, D. R. Richardson: The Evolution of Iron Chelators for the Treatment of Iron Overload Disease and Cancer. Pharmacol. Rev. 2005, 57, 547-583; A. M. Merlot, D. S. Kalinowski, D. R. Richardson: Novel chelators for cancer treatment: where are we now? Antioxid. Redox Signal. 2013, 18, 973-1006; Z. D. Liu, R. C. Hider: Design of iron chelators with therapeutic application Coord. Chem. Rev. 2002, 232, 151-171; H. Nick: Iron chelation, quo vadis? Curr. Opin. Chem. Biol. 2007, 11, 419-423], Another possibility is to affect metalloenzymes, especially ribonucleotide reductase, which plays a key role in the synthesis of DNA as well as other enzymes necessary for the energy metabolism of cells and affecting various cellular processes, such as apoptosis. Based on above mentioned facts, it is evident that chelators can fundamentally affect cell division and growth. Thus, they can serve to overcome resistance in the treatment of cancer.

One of the promising types of chelators are hydrazones. These substances have a wide range of biological activities, including antimicrobial, antimycobacterial, antiviral, fungicidal, antiviral, antimalarial effects or they can serve as therapeutics for the treatment of neurodegenerative diseases. In many cases, hydrazones show significant cytostatic effects [E. A. Malecki, J. R. Connor: The Case for Iron Chelation and/or Antioxidant Therapy in Alzheimer's Disease. Drug Develop. Res. 2002, 56, 526-530; A. Gaeta, R. C. Hider: The crucial role of metal ions in neurodegeneration: the basis for a promising therapeutic strategy. Brit. J. Pharmacol. 2005, 146, 1041-1059; S. Rollas, §. G. Ku^ukguzel: Biological Activities of Hydrazone Derivatives. Molecules 2007, 12, 1910-1939; G. Verma, A. Marella, M. Shaquiquzzaman, M. Akhtar, M. R. Ali, M. M. Alam: A review exploring biological activities of hydrazones. J. Pharm. Bioallied Sci. 2014, 6, 69-80; B. Narasimhan, P. Kumar, D. Sharma: Biological activities of hydrazide derivatives in the new millennium. Acta Pharm. Sci. 2010, 52, 169-180; G. Uppal, S. Bala, S. Kamboj, M. Saini: Therapeutic Review Exploring Antimicrobial Potential of Hydrazones as Promising Lead. Pharma Chem. 2011, 3, 250-268; P. Kumar, B. Narasimhan: Hydrazides/Hydrazones as Antimicrobial and Anticancer Agents in the New Millennium. Mini-Rev. Med. Chem. 2013, 13, 971-987; R. Leon, A. G. Garcia, J. Marco- Contelles: Recent Advances in the Multitarget-Directed Ligands Approach for the Treatment of Alzheimer's Disease. Med. Res. Rev. 2013, 33, 139-189; R. Kaplanek, M. Havlik, B. Dolensky, J. Rak, P. Dzubak, P. Konecny, M. Hajduch, J. Kraiova, V. Kral: Synthesis and biological activity evaluation of hydrazone derivatives based on a Trbger's base skeleton. Bioorg. Med. Chem. 2015, 23, 1651-1659; R. Kaplanek, M. Jakubek, J. Rak, Z. Kejik, M. Havlik, B. Dolensky, I. Frydrych, M. Hajduch, M. Kolar, K. Bogdanova, J. Kraiova, P. Dzubak, V. Kral: Caffeine-hydrazones as anticancer agents with pronounced selectivity toward T- lymphoblastic leukaemia cells. Bioorg. Chem. 2015, 60, 19-29; V. H. Pham, T. P. D. Phan, D. C. Phan, B. D. Vu: Synthesis and Bioactivity of Hydrazide-Hydrazones with the 1-Adamantyl- Carbonyl Moiety. Molecules 2019, 24, 4000; S. Arziman, O. Tanriverdi, S. Kucukvardar, N. Citil, A. Yildiz: Salicylidene acylhydrazides attenuate survival of SH-SY5Y neuroblastoma cells through affecting mitotic regulator Speedy/RINGO and ERK/MAPK-PI3K/AKT signaling. Med. Oncol. 2020, 37, 65; C. Dealwis: Method of modulating ribonucleotide reductase using acylhydrazone analogs to inhibit neoplastic cell growth. PCT Int. Appl., 2017, WO 2017100644 Al; H. Li, L. Chen, R. Tang: Preparation of hexokinase 2 inhibitor compound as antitumor agent. Patent, 2020, CN 111410618 A; D. G. Lloyd, D. Fayne, M. J. Meegan, M. Carr, G. K. Kinsella, L. Caboni, W. N. Jagoe, B. Egan, F. Blanco: Diarylhydrazides for treatment and prevention of androgen-mediated diseases such as prostate cancer, acne, male pattern baldness and hirsutism. PCT Int. Appl., 2013, WO 2013076275 Al; M. Havlik, R. Kaplanek, B. Dolensky, J. Rak, T. Briza, P. Dzubak, M. Hajduch, P. Konecny, J. Stepankova, J. Kraiova, V. Kral: Asymmetric Troger bases with hydrazone group and their use in the treatment of oncologic diseases. Czech Patent, 2015, CZ305683 B6],

Some azulene derivatives also show anticancer effects [T. Wada, R. Maruyama, Y. Irie, M. Hashimoto, H. Wakabayashi, N. Okudaira, Y. Uesawa, H. Kagaya, H. Sakagami: In Vitro Anti-tumor Activity of Azulene Amide Derivatives. In Vivo 2018, 32, 479-486; K. Imanari, M. Hashimoto, H. Wakabayashi, N. Okudaira, K. Bandow, J. Nagai, M. Tomomura, A. Tomomura, Y. Uesawa, H. Sakagami: Quantitative Structure-Cytotoxicity Relationship of Azulene Amide Derivatives. Anticancer Res. 2019, 39, 3507-3518; M. Teratani, S. Nakamura, H. Sakagami, M. Fujise, M. Hashimoto, N. Okudaira, K. Bandow, Y. lijima, J. Nagai, Y. Uesawa, H. Wakabayashi: Antitumor Effects and Tumor-specificity of Guaiazulene-3-Carboxylate Derivatives Against Oral Squamous Cell Carcinoma In Vitro. Anticancer Res. 2020, 40, 4885-4894],

Combination of the azulene and hydrazide-hydrazone pharmacophores leads to hybrid molecules. Azulene hydrazide-hydrazones and the use of these substances as cytostatics for the treatment of oncological diseases are the subject of this patent. of invention

The subject thereof are 7-isopropyl-3-methylazulene-l-carbohydrazones having a substituted 2-hydroxyaryl group (azulene hydrazide-hydrazones) of general formula I,

where R1 R2, R3, R4 are independently H, OH, Cl to C6 alkyl, C(CH₃)₃, allyl, benzyl, phenyl, F, Cl, Br, I, CH₂OH, O(alkyl), CF₃, OCF₃, CN, COO(alkyl), CONH(alkyl), NO₂, N(alkyl)₂, NH(alkyl), NHCO(alkyl), where (alkyl) are alkyl Cl to C6, or R1-R2 or R2-R3 or R3-R4 is -CH=CH-CH=CH-, i.e., condensed benzene ring.

The compounds of general formula I have cytostatic effects and thus they can be used for the preparation of therapeutic systems for the treatment of oncological diseases.

Preparation of azulene hydrazide-hydrazones with 2-hydroxyaryl group of general formula I and their properties are documented in examples below without being limited thereto.

Brief description of the Figures

Figure 1 shows structures of chelators 13-15

Figure 2 shows titration curves for titration of the chelator 13 with ferrous and ferric ions

Figure 3 shows titration curves for titration of the chelator 14 with ferrous and ferric ions

Figure 4 shows titration curves for titration of the chelator 15 with ferrous and ferric ions

Figure 5 shows intracellular localization of chelators 13 (A), 14 (B) and 15 (C) in living HF cells.

1) chelators [1 pM]; 2) LysoTracker™ [300 nM]; 3) MitoTracker™ [50 nm]; 4) merge

Figure 6 shows upregulation of NDRG1, TfRl and HIF-l-alfa for control, DFO [100 pM],

Dp44mT [5 pM], Dpc [5 pM] and chelators 13, 14 and 15 [5 pM], Example 1. Preparation of starting material

Preparation of azulene ester: 5-lsopropyl-3-(methoxycarbonyl)-2H-cyclohepta[b]furan-2-on (1232 mg; 5 mmol) was dissolved in isopropanol (16 mL). To the solution was then added propionaldehyde (1.1 mL; 15 mmol) followed by morpholine (1.35 mL; 15.3 mmol). Reaction mixture was stirred at 75 °C for 4 h (TLC: petroleum ether/ethyl acetate 8:1 v/v). Then volatile compounds were evaporated under reduced pressure and crude product was purified by column chromatography on silica (eluent petroleum ether/ethyl acetate 5:1 v/v). It was obtained 975 mg (yield 80%) of methyl 7-isopropyl-3-methylazulene-l-carboxylate in the form of dark blue solid.

^XH NMR (300 MHz; CDCI₃) 1.41 (d, J = 6.9 Hz, 6H); 2.58 (s, 3H); 3.20 (hept, J = 6.9 Hz, 1H); 3.94 (s, 1H); 7.34 (m, 1H); 7.69 (d, J = 10.2 Hz, 1H); 8.16 (s, 1H); 8.23 (dd, J = 9.8, 0.6 Hz, 1H); 9.67 (d, J = 1.9 Hz, 1H), ¹³C NMR (75 MHz, CDCI3) 12.6; 24.8; 39.2; 51.1; 113.5; 124.3; 125.3; 133.6; 137.1; 137.8; 140.8; 141.2; 141.6; 148.2; 166.1

Preparation of 7-isopropyl-3-methylazulene-l-carbohydrazide: Methyl 7-isopropyl-3- methylazulene-l-carboxylate (952 mg; 3.93 mmol) was dissolved in isopropanol (30 mL). Then hydrazin hydrate (10 mL; 200 mmol) was added and reaction mixture was stirred at 75 °C overnight. Then volatile compounds were evaporated under reduced pressure and crude product was purified by column chromatography on silica (eluent ethyl acetate). It was obtained 876 mg (yield 92%) of 7-isopropyl-3-methylazulene-l-carbohydrazide in the form of dark blue solid.

^TH NMR (300 MHz; CDCI3) 1.39 (d, J = 6.9 Hz, 6H); 2.57 (s, 3H); 3.18 (hept, J = 6.9 Hz, 1H); 4.20 (s, 2H); 7.29 (m, 1H); 7.39 (s, 1H); 7.67 (m, 1H); 7.79 (s, 1H); 8.21 (dd, J = 9.9, 1.0 Hz, 1H); 9.66 (d, J = 1.9 Hz, 1H), ¹³C NMR (75 MHz; CDCI3) 12.6; 24.8; 39.1; 115.2; 124.1; 124.5; 133.8; 135.7; 137.8; 138.1; 140.0; 140.3; 147.3; 167.7 Example 2. Preparation of azulene hydrazide-hydrazones of general formula I.

General method. 7-isopropyl-3-methylazulene-l-carbohydrazide (46 mg; 0.19 mmol) and substituted 2-hydroxybenzaldehyde (0.20 mmol) were dissolved in isopropanol (10 mL). Reaction mixture was stirred at 75 °C for 2 days. Then volatile compounds were evaporated under reduced pressure and crude product was suspended in the mixture of diethyl ether / petroleum ether (1:1 v/v; 30 mL). Solid product was filtered of on the frit S4, washed with next portion of diethyl ether / petroleum ether mixture (1:1 v/v; 3x 20 mL) and dried under vacuum at 50 °C. Yields was 60-97%.

Examples of prepared compounds including their structures, structures of starting 2- hydroxybenzaldehydes, preparative yields and ¹H NMR of azulene hydrazide-hydrazones are summarized in Table 1.

Table 1. Structures, yields and ¹H NMR of azulene hydrazide-hydrazones

Example 3. Chelating properties of azulene hydrazide-hydrazones toward ferrous and ferric ions.

Chelation of iron ions is one of the possible mechanisms of action hydrazide-hydrazones. Therefore, tests of chelating properties of selected azulene hydrazide-hydrazones toward ferrous and ferric ions in aqueous medium (water-DMSO 99: 1, v/v) were performed. The binding constants are significantly affected by the solvent used, so all experiments were performed in the same system (water-DMSO). UV/Vis titration of /V'-(2-hydroxy-3- methoxybenzylidene)-7-isopropyl-3-methylazulene-l-carbohydrazide (chelator 13), /V'-((2- hydroxynaphthalen-l-yl)methylen)-7-isopropyl-3-methylazulene-l-carbohydrazide (chelator 14) and /V'-(4-(diethylamino)-2-hydroxybenzylidene)-7-isopropyl-3-methylazulene-l- carbohydrazide (chelator 15), all of general formula I with Fe²⁺ and Fe³⁺ ions showed, that all compounds have strong affinity to these ions (there were strong spectral responses after addition of iron ions). Chelator's concentration was 10 pM, Fe²⁺ or Fe³⁺ concentration was in the range of 0-0.5 mM. UV spectra were recorded in the range of 300-600 nm.

On the figures 2-4 are shown titration curves for titration of chelators 13-15 with ferrous and ferric ions.

Example 4. Anticancer effect of azulene hydrazide-hydrazones.

Cytotoxic effect of azulene hydrazide-hydrazones were evaluated on different pancreatic cell lines: MIA PaCa and PANC-1; HF (human fibroblasts) were used as control. In the tables 2 and 3 are shown structures of tested compounds and summarized their IC₅₀ values (as pM or pg/mL). IC₅o values were measured after 48h incubation.

The cytotoxicity values (IC₅₀) for tested compounds were in the range of 0.195 to 2.073 pM (0.07 pg/mL to 0.87 pg/mL). The most effective cytotoxic effect was observed on MIA-PaCa cells for /V'-((2-hydroxynaphthalene-l-yl)methylene)-7-isopropyl-3-methylazulene- 1-carbohydrazide (chelator 14). From the measured data is clear that chelators have a great cytotoxic potential. Table 2. IC₅₀ [M] for selected azulene hydrazide-hydrazones

Table 3. IC₅₀ [pg/mL] for selected azulene hydrazide-hydrazones

Example 5. In vitro imaging

For testing the intracellular localization was used HF (human fibroblasts) cells, HF were seeded in numbers of 5,000 cells per well. The cells were left to adhere at least 24 hours in incubator. After incubation, cells were washed two times with PBS. Then was added complete cell culture medium (DMEM) with chelators in concentration of 1 pM, MitoTracker™ Red in concentration of 50nM and LysoTracker™ Green in concentration of 300 nM. After adding the mixture, cells were incubated for 15 minutes (37°C, 5% CO₂), intracellular uptake of the chelators was quite fast. After incubation the cells were washed two times with PBS and medium without phenol red was added. A fluorescence real-time microscopy study on live cells was performed on Leica TCS SP8 WLL SMD-FLIM microscope in atmosphere with 5% CO₂, at 37°C. For visualization of cells was used a water objective HC PL APO CS2 63x (NA1,2) and three lasers with different excitation wavelengths: 1. 405 nm (10% laser power), 2. 504 nm (8% laser power), 3. 579 nm (8% laser power), it was used HyD detectors (400 - 720 nm). Intracellular localization (Fig. 5.) of chelators was compared with localizations of MitoTracker™ and LysoTracker™. It is obvious, that the chelators can cross cell membrane quickly and are mainly concentrating in lysosomes.

Example 6. Western Blot

The changes of proteins (NDRG1, TfRl, Hif-l-alpha) expression were studied on AsPCl tumor cell lines, after incubation with iron chelators. The investigation of the effect of iron chelators on protein expression was based on comparing our newly synthesized chelators (5 pM) with commercial chelators - DFO (100 p.M), Dpc (5 pM) and DP44mT (5 pM). AsPCl was seeded in number of 600,000 cells per well and were left to adhere at least for 24 hours, till confluency was 70%. It was used RPMI 1640 medium (20% FBS, 1% streptomycin). After incubation was removed all medium and mixture of medium and chelator was added. It was left incubated for 24 hours. After 24 hours cell lyses was done. From cell lysate was determinate increasing or decreasing effect on protein in cells in comparison with control (cells without chelator). For tested proteins there are three replications of experiments available to verify the result. The proteins expression changing was tested for three proteins (NDRG1, TfRl, Hif-l-a Ipha), whose are important for cancer cell lines (Fig.6). TfRl (transferrin receptor) is protein involved in cellular iron uptake as well as in regulation of cell growth. The expression of TfR is mainly regulated at the post-transcriptional level and its response to intracellular iron level. NDRG1 (N-myc downstream regulated protein) is iron regulated metastasis suppressor protein. It is a protein involved mainly in cell growth, differentiation, DNA reparation as well as cell adhesion. HiF-1- alpha (hypoxia-inducible factor-l-alpha) is important in tumour angiogenesis. It is activated by low oxygen and by genetic events connected with tumour disease. Under hypoxia HiF-l-alpha becomes stable and acts as important regulator of numerous hypoxia inducible genes, whose are related to angiogenesis, cell proliferation and glucose/ iron metabolism.

Claims

Claims

1. Azulene hydrazide-hydrazones having 2-hydroxyaryl group of general formula I,

where R1 R2, R3, R4 are independently H, OH, Cl to C6 alkyl, C(CH₃)₃, allyl, benzyl, phenyl, F, Cl, Br, I, CH₂OH, O(alkyl), CF₃, OCF₃, CN, COO(alkyl), CONH(alkyl), NO₂, N(alkyl)₂, NH(alkyl), NHCO(alkyl), where (alkyl) are alkyl Cl to C6, or R1-R2 or R2-R3 or R3-R4 is -CH=CH-CH=CH-, i.e., condensed benzene ring.

2. Use of compounds of general formula I according to claim 1 for preparation of a medicament for the treatment of oncological diseases.