N-aminoguanidine derivatives with anticancer activity

Abstract

N-Aminoguanidine derivatives according to Formula (1), pharmaceutical compositions containing them and their use in the treatment of cancer.

Description

[0001] N-AMINOGUANIDINE DERIVATIVES WITH ANTICANCER ACTIVITY

[0002] Field of the invention

[0003] The present invention relates to new N-aminoguanidine derivatives, a process of their preparation and their use in the treatment of cancer.

[0004] Background of the invention

[0005] Cancer refers to a group of diseases with a high mortality rate, in which abnormal cells divide without control and invade surrounding tissues. Despite novel cancer therapies that have emerged, chemotherapy remains the most common method for cancer treatment. This systemic type of treatment involves the administration of powerful drugs designed to eliminate rapidly dividing cancer cells throughout the body. Depending on their chemical structures and the way they act on cancer cells, chemotherapeutic agents can be classified into various categories such as antimetabolites, alkylating agents, platinum analogs, vinca alkaloids, anthracyclines, etc. Platinum chemotherapy is one of the oldest and most widely used cancer treatments until today. Platinumbased chemotherapeutics (cisplatin, oxaliplatin, carboplatin) exhibit their anticancer activity by interfering with crucial cellular processes combining cytotoxic and cytostatic properties.

[0006] While one of the main purposes of chemotherapy is selectivity in terms of targeting and destroying rapidly dividing cancer cells, it is usually inevitable not to harm healthy cells, leading to a variety of distressing side effects including nausea, vomiting, hair loss, neurotoxicity and nephrotoxicity. In addition, an ever-evolving problem is that despite initial successes, many cancers display a remarkable ability to adapt and evolve, rendering traditional chemotherapeutic agents ineffective over time. The development of chemotherapy resistance has led to the need for novel, more potent anti-tumor agents and chemotherapeutic approaches, however, characterized by greater selectivity.

[0007] Azine, guanidine, and N-heterocyclic carbene (NHC)-based compounds have been shown to exert diverse chemical, biological, and physical properties ((1) Chourasiya, S. S.; Kathuria, D.; Wani, A. A.; Bharatam, P. V. Azines: Synthesis, Structure, Electronic Structure and Their Applications. Org. Biomol. Chem. 2019, 17 (37), 8486- 8521.; (2) Gomes, A. R.; Varela, C. L.; Pires, A. S.; Tavares-da-Silva, E. J.; Roleira, F. M. F. Synthetic and Natural Guanidine Derivatives as Antitumor and Antimicrobial Agents: A Review. Bioorg. Chem. 2023, 138, 106600.; (3) Guo, R.; Zheng, Y.; Ma, J.- A. Electrophilic Reaction of 2,2,2-Trifluorodiazoethane with the in Situ Generated N- Heterocyclic Carbenes: Access to N-Aminoguanidines. Org. Lett. 2016, 18 (17), 4170- 4173; (4) Basu, A.; Sinha, B. N.; Saiko, P.; Szekeres, T. Effect of Substitution at N"- Position of N'-Hydroxy-N-Amino Guanidines on Tumor Cell Growth. Bioorg. Med. Chem. Lett. 2012, 22 (15), 4934-4938; (5) Costas, M.; Vila, N.; Rahman, A.; Besada, P.; Rozas, I.; Brea, J.; Loza, M.; Gonzalez-Romero, E.; Teran, C. Novel Pyridazin-3(2 H )-One-Based Guanidine Derivatives as Potential DNA Minor Groove Binders with Anticancer Activity. ACS Med. Chem. Lett. 2022, 73; (6) Vougioukalakis, G. C.; Grubbs, R. H. Chem. Rev. 2010, 110, 1746-1787. “Ruthenium-Based Heterocyclic Carbene- Coordinated Olefin Metathesis Catalysts”).

[0008] Summary of the invention

[0009] The present invention provides novel N-aminoguanidine derivatives which exhibit anticancer activity.

[0010] Specifically, the present invention provides a compound of Formula (1) or a pharmaceutically acceptable salt thereof, wherein

[0011] = = = is a single bond, or a double bond, or no bond, wherein when = = = is single bond, n is 1 , 2 or 3, when = = = is double bond, n is 1 when = = = is no bond, n is 1 ;

[0012] Ri and R2 are the same or different and are selected from the group consisting of H, C1-C15 alkyl, CH2-phenyl, cycloalkyl, heteroaryl and aryl, wherein R1 and R2 are optionally substituted with one or more substituents which are the same or different and are selected from the group consisting of C1-C15 alkyl, C1-C15 alkoxy, NH2, NO2,

[0013] CN, CF3, Cl, Br and I; Rs and R4 are the same or different and are selected from the group consisting of C1- C15 alkyl, CHs-phenyl, cycloalkyl, heteroaryl and aryl, wherein R3 and R4 are optionally substituted with one or more substituents which are the same or different and are selected from the group consisting of C1-C15 alkyl, C1-C15 alkoxy, NH2, NO2, CN, CF3, Cl, Br and I;

[0014] Rs is selected from the group consisting of H, C1-C15 alkyl, CHs-phenyl, heteroaryl and aryl, wherein Rs is optionally substituted with one or more substituents, which are the same or different and are selected from the group consisting of C1-C15 alkyl, C1-C15 alkoxy, heteroaryl or aryl, NH2, NO2, CN, CF3, F, Cl, Br and I; provided that the compound of Formula (1) is not

[0015] The present invention provides also a compound as defined above for use in the treatment of cancer.

[0016] The present invention provides also a pharmaceutical composition comprising as active ingredient a compound of as defined above and a pharmaceutically acceptable carrier.

[0017] Brief description of the drawings

[0018] Figure 1 shows the cytotoxic activity of compounds of the present invention against A2780 ovarian cancer cells.

[0019] Figure 2 shows the cytotoxic activity of compounds of the present invention against A2780 ovarian cancer cells in comparison to oxaliplatin.

[0020] Figure 3 shows the cytotoxic activity of compounds of the present invention against MDA-MB-231 breast cancer cells in comparison to oxaliplatin.

[0021] Figure 4 shows the cytotoxic activity of compounds of the present invention against HT29 colorectal adenocarcinoma cells in comparison to oxaliplatin. Figure 5 shows dose-response curves of compounds of the present invention against malignant cell lines A2780, SKOV3 and HeLa.

[0022] Figure 6 shows dose-response curves of compounds of the present invention against malignant cell lines HT29, MDA-MB-231 and VmCubl .

[0023] Figure 7 shows the apoptotic effects of a compound of the present invention on A2780 ovarian cancer cells.

[0024] Figure 8 shows the effects of a compound of the present invention on early and late apoptosis of A2780 ovarian cancer cells.

[0025] Figure 9 shows the results of flow cytometric analysis after treatment of A2780 ovarian cancer cells with a compound of the present invention.

[0026] Detailed description of the invention

[0027] The present invention provides a compound of Formula (1) or a pharmaceutically acceptable salt thereof, wherein

[0028] = = = is a single bond, or a double bond, or no bond, wherein when = = = is single bond, n is 1 , 2 or 3, when = = = is double bond, n is 1 when = = = is no bond, n is 1 ;

[0029] Ri and R2 are the same or different and are selected from the group consisting of H, C1-C15 alkyl, CH2-phenyl, cycloalkyl, heteroaryl and aryl, wherein R1 and R2 are optionally substituted with one or more substituents which are the same or different and are selected from the group consisting of C1-C15 alkyl, C1-C15 alkoxy, NH2, NO2, CN, CF3, Cl, Br and I; R3 and R4 are the same or different and are selected from the group consisting of C1- C15 alkyl, CH2-phenyl, cycloalkyl, heteroaryl and aryl, wherein R3 and R4 are optionally substituted with one or more substituents which are the same or different and are selected from the group consisting of C1-C15 alkyl, C1-C15 alkoxy, NH2, NO2, CN, CF3, Cl, Br and I;

[0030] Rs is selected from the group consisting of H, C1-C15 alkyl, CH2-phenyl, heteroaryl and aryl, wherein Rs is optionally substituted with one or more substituents, which are the same or different and are selected from the group consisting of C1-C15 alkyl, C1-C15 alkoxy, heteroaryl or aryl, NH2, NO2, CN, CF3, F, Cl, Br and I; provided that the compound is not

[0031] Preferably Ri and R2 are the same or different and are selected from H, or C1-C4 alkyl. More preferably, R1 and R2 are the same or different and are selected from a C1-C4 alkyl. Even more preferably, both R1 and R2 are methyl groups.

[0032] Preferably, R3 and R4 are selected from the group consisting of C1-C4 alkyl, CH2- phenyl, heteroaryl, such us pyridyl, and aryl. More preferably, R3 and R4 are heteroaryl, or aryl. Even more preferably, R3 and R4 are 1 ,3,5-trimethylphenyl (mesityl), or 2,6 - diisopropylphenyl. Most preferably, R3 and R4 are the same and are mesityl, or 2,6 - diisopropylphenyl.

[0033] Rs is preferably selected from the group consisting of C1-C4 alkyl, benzyl, heteroaryl and aryl. More preferably, Rs is a tert-butyl group.

[0034] Preferably, = = = is a double bond and n is 1.

[0035] Preferred compounds according to the present invention are the following:

[0036] (E)-benzyl 2-((1 ,3-dicyclohexyl-1 H-imidazol-2(3H)-ylidene)hydrazono)acetate;

[0037] (E)-benzyl 2-((1 , 3-dimesityl- 1 H-imidazol-2(3H)-ylidene)hydrazono)acetate;

[0038] (E)-benzyl 2-((bis(mesityl(methyl)amino)methylene)hydrazono)acetate; (E)-benzyl 2-((1 ,3-dimesityltetrahydropyrimidin-2(1 H)-ylidene)hydrazono)acetate;

[0039] (E)-tert-butyl 2-((1 ,3-bis(2,6-diisopropylphenyl)-4,5-dimethyl-1 H-imidazol-2(3H)- ylidene)hydrazono)acetate;

[0040] (E)-benzyl 2-((1 , 3-dimesityl- 1 ,3-diazepan-2-ylidene)hydrazono)acetate;

[0041] (E)-benzyl 2-((1 ,3-dimesitylimidazolidin-2-ylidene)hydrazono)acetate;

[0042] (E)-ethyl 2-((1 ,3-dimesitylimidazolidin-2-ylidene)hydrazono)acetate;

[0043] (E)-ethyl 2-((1 ,3-bis(2,6-diisopropylphenyl)imidazolidin-2-ylidene)hydrazono)acetate;

[0044] (E)-methyl 2-((1 , 3-di mesityl- 1 H-imidazol-2(3H)-ylidene)hydrazono)acetate;

[0045] (E)-tert-butyl 2-((1 ,3-bis(2,6-diisopropylphenyl)tetrahydropyrimidin-2(1 H)- ylidene)hydrazono)acetate;

[0046] (E)-tert-butyl 2-((1 ,3-bis(2,6-diisopropylphenyl)-1 ,3-diazepan-

[0047] 2ylidene)hydrazono)acetate;

[0048] (E)-tert-butyl 2-((1 ,3-dimesityl-4,5-dimethyl-1 H-imidazol-

[0049] 2(3H)ylidene)hydrazono)acetate.

[0050] The compounds of the present invention contain at least one basic functional group, which means that they can form an acid addition salt by treatment with a suitable acid. Suitable acids include pharmaceutically acceptable inorganic and pharmaceutically acceptable organic acids. Examples of pharmaceutically acceptable acid addition salts include hydrochloride, hydrobromide, sulfate, phosphate, nitrate, acetate, propionate, butyrate, maleate, fumarate, tartrate, citrate, lactate, oxalate, succinate, and benzoate.

[0051] The compounds of the present invention may also contain an acidic functional group, for example, when Rs is hydrogen. In such a case, the compounds can form a base addition salt by treatment with a suitable base. Examples of base addition salts include ammonium, sodium, potassium, magnesium, calcium salts and salts with amino acids, for example arginine, or lysine.

[0052] The pharmaceutically acceptable salts can be prepared by using methods well known in the art.

[0053] A general route of synthesis of the compounds of Formula (1) is presented below in Scheme 1. (ij (ii) x SeN''N R

[0054] R / +D Se JN5y-R5 3"N N'^4Base R3 - N^N 'R4

[0055] - triphenylphosphine

[0056] Ri R2 Ri R'

[0057] (A) (B)

[0058] Scheme 1

[0059] The starting compound is an azolium salts (A). Azolium salts and their processes of preparation are well known in the art (N-Heterocyclic Carbenes, 1st ed.; Nolan, S. P., Ed.; Wiley, 2014; G. C. Vougioukalakis, R. H. Grubbs. Chem. Rev. 2010 110, 1746- 1787).

[0060] In step (i), an azolium salt (A) is treated with Se and a base in an organic solvent, under heating, to give the corresponding selenourea (B). In step (ii), the selenourea (B) is treated with the appropriate diazo compound and triphenylphosphine in a suitable solvent under heating.

[0061] For example, step (i) can be performed by treating 1 equivalent of compound (A) with 1.1 equivalent of selenium and 3 equivalents of potassium carbonate in acetone under reflux for 16 hours. The solvent is then evaporated and the residue is filtered through a silica gel plug using dichloromethane. After removing dichloromethane under vacuum, compound (B) is isolated as a solid. No further purification is required. Step (ii) can be for example performed by treating compound (B) with 3 equivalents triphenylphosphine and 1 .2 equivalents of the diazo compound in toluene. The reaction mixture is stirred at 110 °C for 16 h. The solvent is then removed under vacuum and the residue is filtered through a silica gel plug with diethyl ether. Flash column chromatography on silica gel (pentane I diethyl ether) affords pure compound (1).

[0062] The compounds of the present invention exhibit strong anticancer activity. Examples of cancers which can be treated with the compounds of the present invention include ovarian cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, liver cancer, breast cancer, and melanoma.

[0063] A compound of Formula (1) of a pharmaceutically acceptable salt thereof is typically administered to a subject as a pharmaceutical composition. Preferably, the subject is a human. Thus, the present invention provides also a pharmaceutical composition comprising as active ingredient a compound of Formula (1) or a pharmaceutically acceptable salt thereof, as defined above.

[0064] The composition may be formulated, for example, for oral, parenteral, intramuscular, intravenous, intraperitoneal, subcutaneous, transdermal / intradermal, or inhalation administration. The composition may have different forms, such as solid or liquid forms, for example it can have the form of powder, tablet, capsule, suppository, solution, suspension, emulsion, gel, cream, ointment, spray, or transdermal patch.

[0065] The composition typically comprises a compound of Formula (1), or a pharmaceutically acceptable salt thereof as active ingredient and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may comprise one or more excipients, such as diluents, binders, disintegrants, lubricants, antioxidants, solvents, buffering agents, preservatives, or emulsifying agents, which are well known in the art.

[0066] Examples of diluents include starch derivatives, such as corn starch, potato starch or rice starch, polysaccharides such as dextrins, maltodextrins, dextrates, microcrystalline cellulose, powdered cellulose, lactose, sucrose, dextrose, mannitol and sorbitol.

[0067] Examples of binders include carboxymethylcellulose, or a salt thereof, such as calcium, or sodium salt, ethyl cellulose hydroxypropylcellulose, hydroxyethylcellulose, povidone, sodium alginate, dextrose, xylitol, polyvinylpyrrolidone and polyethylene glycol.

[0068] Examples of disintegrants include alginic acid, carboxymethylcellulose, or a salt thereof, such as calcium or sodium salt, microcrystalline cellulose, croscarmellose sodium, crospovidone, sodium docusate, hydroxypropyl cellulose, povidone, sodium alginate, sodium starch glycolate, starch and pregelatinized starch.

[0069] Examples of lubricants include magnesium stearate, stearic acid, sodium stearyl fumarate, sodium lauryl sulphate, magnesium lauryl sulphate, and talc.

[0070] Examples of antioxidants include sodium metabisulfite, butylated hydroxyanisole, butylated hydroxytoluene and ascorbic acid.

[0071] Examples of solvents, include saline, water, phosphate buffered saline, ethanol, isopropyl alcohol, and mixtures thereof. Examples of buffering agents include citric acid monohydrate, acetic acid, sodium acetate, and sodium hydrogen phosphate.

[0072] Examples of preservatives include sodium benzoate, benzyl alcohol and parahydroxy benzoic acids and their alkyl esters.

[0073] Examples of emulsifying agents include sodium lauryl sulfate, lecithin, polysorbate and sorbitan monooleate.

[0074] The composition of the present invention can be administered, for example, orally, parenterally, intramuscularly, intravenously, subcutaneously, transdermally, intraperitoneally, or by inhalation. The route of administration will depend on various factors, such as the nature, location and severity of the disease to be treated.

[0075] The pharmaceutical composition is generally administered daily, or more frequently, such as twice a day, or less frequently, such as every second day, or every week. The frequency of administration and the administered dose depend on various factors, such as the disease, the route of administration, the age of the subject, and the form of the administered composition, and can be determined by a person skilled in the art by using common general knowledge.

[0076] This example shows the preparation of compounds according to the present invention. The general synthetic procedure for the preparation of the compounds is shown in Scheme 2.

[0077] Scheme 2 (i) In a vial equipped with a stirring barwere added the azolium salt (A) (2 mmol), K2CO3 (6 mmol, 3 equiv.) and selenium (0.470 g, 6 mmol, 3 equiv.) in acetone (10 mL). The reaction mixture was stirred at 60 °C for 16 h. The solvent was evaporated and the residue was filtered through a silica gel plug using dichloromethane. After removing dichloromethane under vacuum, the / V-heterocyclic carbene-based (NHC) selenourea (B) was isolated as a solid. No further purification was required.

[0078] (ii) / V-heterocyclic carbene-based (NHC) selenourea B (0.22 mmol), PPhs (0.66 mmol, 3 equiv.), and diazo compound (0.26 mmol, 1.2 equiv.) in toluene (0.9 mL) were added into a 4 mL vial, equipped with a stirring bar. The reaction mixture was stirred at 110 °C for 16 h. The solvent was then removed under vacuum and the residue was filtered through a silica gel plug with diethyl ether. Flash column chromatography on silica gel (pentane I diethyl ether) afforded the pure product.

[0079] (E)-benzyl 2-((1 ,3-dicyclohexyl-1 H-imidazol-2(3H)-ylidene)hydrazono)acetate

[0080] (EZT190)

[0081] Synthesized according to the general procedure, purified by column chromatography (diethyl ether / pentane) as a yellow solid, isolated yield 90%.

[0082] 1H NMR (200 MHz, CDCI3) 6 7.53 - 7.22 (m, 6H), 6.50 (s, 2H), 5.25 (s, 2H), 4.73 (m, 2H), 2.33 - 0.94 (m, 20H).

[0083] 13C NMR (125 MHz, CDCI3) 6 166.3, 150.3, 137.0, 132.1 , 129.2, 128.4, 127.9, 111.2, 65.5, 55.1 , 33.0, 25.4, 25.4.

[0084] HRMS (ESI) m / z cal. for (M + H)+409.2598, found 409.2660.

[0085] (E)-benzyl 2-((1 ,3-dimesityl-1 H-imidazol-2(3H)-ylidene)hydrazono)acetate (EZT197)

[0086] Synthesized according to the general procedure, purified by column chromatography (diethyl ether / pentane) as a yellow solid, isolated yield 96%.

[0087] 1H NMR (400 MHz, CDCI3) 6 7.40 - 7.31 (m, 3H), 7.28 - 7.22 (m, 2H), 7.16 (s, 1 H), 6.86 (s, 4H), 6.42 (s, 2H), 4.96 (s, 2H), 2.28 (s, 6H), 2.16 (s, 12H).

[0088] 13C NMR (100 MHz, CDCI3) 6 165.9, 151.5, 138.1 , 137.3, 135.5, 134.1 , 133.6, 128.97, 128.3, 127.7, 127.6, 116.3, 64.9, 21.2, 18.1.

[0089] HRMS (ESI) m / z cal. for (M + H)+481.2598, found 481.2679.

[0090] Synthesized according to the general procedure, purified by column chromatography (diethyl ether / pentane) as a yellow solid, isolated yield 60%.

[0091] 1H NMR (200 MHz, CDCI3) 6 7.72 (s, 1 H), 7.47 - 7.24 (m, 5H), 6.68 (s, 4H), 5.30 (s, 2H), 3.10 (s, 6H), 2.23 (s, 6H), 1.76 (s, 12H).

[0092] 13C NMR (50 MHz, CDCI3) 6 166.5, 165.3, 140.0, 137.4, 137.0, 136.5, 136.2, 129.5, 128.6, 128.0, 127.9, 66.0, 40.8, 20.9, 17.2.

[0093] HRMS (ESI) m / z cal. for (M + H)+485.2911 , found 485.2986. (E)-benzyl 2-((1 ,3-dimesityltetrahydropyrimidin-2(1 H)-ylidene)hydrazono)acetate

[0094] (EZT243)

[0095] Synthesized according to the general procedure, purified by column chromatography (diethyl ether I pentane) as a yellow solid, isolated yield 66%.

[0096] 1H NMR (400 MHz CDCI3) 6 7.42 - 7.26 (m, 5H), 6.83 (s, 1 H), 6.76 (s, 4H), 4.95 (s, 2H), 3.54 (t, J = 6.0 Hz, 4H), 2.28 - 2.20 (m, 20H).

[0097] 13C NMR (100 MHz, CDCI3) 6 165.5, 156.5, 141.0, 136.8, 135.9, 134.5, 133.9, 129.0, 128.3, 128.3, 127.8, 65.3, 48.8, 23.7, 21.0, 18.5. HRMS (ESI) m / z cal. for (M + H)+497.2911 , found 497.2955.

[0098] (E)-tert-butyl 2-((1 ,3-bis(2,6-diisopropylphenyl)-4,5-dimethyl-1 H-imidazol-2(3H)- ylidene)hydrazono)acetate (EZT230)

[0099] Synthesized according to the general procedure, purified by column chromatography (diethyl ether / pentane) as a yellow solid, isolated yield 83%.

[0100] 1H NMR (200 MHz, CDCI3) 6 7.47 - 7.11 (m, 6H), 6.90 (s, 1 H), 2.78 (hept, J = 6.7 Hz, 4H), 1 .72 (s, 6H), 1.37 - 0.98 (m, 33H).

[0101] 13C NMR (50 MHz, CDCI3) 6 166.2, 151.7, 146.5, 135.9, 132.4, 129.5, 123.8, 118.5,

[0102] 79.1 , 29.1 , 28.5, 23.8, 23.5, 9.4. HRMS (ESI) m / z cal. for (M + H)+559.4007, found 559.4103.

[0103] (E)-benzyl 2-((1,3-dimesityl-1,3-diazepan-2-ylidene)hydrazono)acetate (EZT249)

[0104] Synthesized according to the general procedure, purified by column chromatography (diethyl ether / pentane) as an orange solid, isolated yield 71%.

[0105] 1H NMR (200 MHz CDCI3) 6 7.51 - 7.17 (m, 5H), 6.79 (s, 1 H), 6.72 (s, 4H), 4.98 (s, 2H), 4.01 - 3.33 (m, 4H), 2.32 - 2.13 (m, 18H), 1.80 - 1.58 (m, 4H).

[0106] 13C NMR (50 MHz, CDCI3) 6 164.9, 159.5, 143.1, 137.8, 136.6, 135.5, 134.9, 129.4, 128.7, 128.6, 128.2, 65.9, 55.1 , 28.2, 21.0, 20.2. HRMS (ESI) m / z cal. for (M + H)+511.3068, found 511.3109.

[0107] (E)-benzyl 2-((1,3-dimesitylimidazolidin-2-ylidene)hydrazono)acetate (EZT204)

[0108] Synthesized according to the general procedure, purified by column chromatography (diethyl ether I pentane) as a yellow solid, isolated yield 54% (isolated yield 90% when 2 equiv. of diazo compound were used).

[0109] 1H NMR (400 MHz, CDCI3) 6 7.47 - 7.24 (m, 5H), 7.17 (s, 1H), 6.85 - 6.81 (m, 4H), 4.96 (s, 2H), 3.86 (s, 4H), 2.36 - 2.24 (m, 18H).13C NMR (100 MHz, CDCI3) 6 165.3, 159.3, 137.3, 136.8, 136.0, 129.8, 129.6, 129.2, 128.3, 127.9, 127.8, 65.4, 47.9, 21.1 , 18.1.

[0110] HRMS (ESI) m / z cal. for (M + H)+483.2755, found 483.2840.

[0111] (E)-ethyl 2-((1 ,3-dimesitylimidazolidin-2-ylidene)hydrazono)acetate (EZT201)

[0112] Synthesized according to the general procedure, purified by column chromatography (diethyl ether I pentane) as a yellow solid, isolated yield 62% (isolated yield 89% when 2 equiv. of diazo compound were used).

[0113] 1H NMR (400 MHz, CDCI3) 6 7.10 (s, 1 H), 6.91 (s, 4H), 3.94 (q, J = 7.1 Hz, 2H), 3.84 (s, 4H), 2.30 (d, J = 5.1 Hz, 18H), 1.13 (t, J = 7.1 Hz, 3H).

[0114] 13C NMR (100 MHz, CDCI3) 6 165.4, 158.8, 137.8, 129.8, 129.7, 129.5, 129.2, 59.7, 47.2, 21.0, 18.1 , 14.1.

[0115] HRMS (ESI) m / z cal. for (M + H)+421.2598, found 421.2695.

[0116] (E)-ethyl 2-((1 ,3-bis(2,6-diisopropylphenyl)imidazolidin-2-ylidene)hydrazono)acetate (EZT228)

[0117] Synthesized according to the general procedure, purified by column chromatography (diethyl ether I pentane) as a yellow solid, isolated yield 87% when 2 equiv. of diazo compound were used.1H NMR (200 MHz, CDCI3) 6 7.38 - 7.08 (m, 6H), 7.02 (s, 1 H), 4.00 - 3.77 (m, 6H), 3.17 (hept, J = 7.0 Hz, 4H), 1.33 (d, J = 6.9 Hz, 12H), 1.23 - 1.15 (d, J = 6.9 Hz, 12H), 1.04 (t, J = 7.1 Hz, 3H).

[0118] 13C NMR (126 MHz, CDCI3) 6 165.6, 159.9, 147.6, 145.6, 137.9, 128.7, 127.9, 124.0, 59.5, 51.5, 49.1 , 29.1 , 24.2, 14.4.

[0119] HRMS (ESI) m / z cal. for (M + H)+505.3537, found 505.3563.

[0120] (E)-methyl 2-((1 ,3-dimesityl-1 H-imidazol-2(3H)-ylidene)hydrazono)acetate (EZT278)

[0121] Synthesized according to the general procedure, purified by column chromatography (diethyl ether / pentane) as a yellow solid, isolated yield 79%.

[0122] 1H NMR (200 MHz, CDCI3) 6 7.07 (s, 1 H), 6.94 (s, 4H), 6.42 (s, 2H), 3.42 (s, 3H), 2.33 (s, 8H), 2.15 (s, 12H).

[0123] 13C NMR (50 MHz, CDCI3) 6 166.5, 151.4, 138.2, 135.5, 134.1 , 133.3, 128.9, 116.3, 50.7, 21.2, 18.1.

[0124] HRMS (ESI) m / z cal. for (M + H)+505.3537, found 505.3563.

[0125] (E)-tert-butyl _ 2-((1 ,3-bis(2,6-diisopropylphenyl)tetrahydropyrimidin-2(1 H)- ylidene)hydrazono)acetate (EZT350)

[0126] Synthesized according to the general procedure, purified by column chromatography (diethyl ether / pentane) as a yellow solid, isolated yield 69%.1H NMR (500 MHz, CDCI3) 6 7.25 - 7.20 (m, 2H), 7.11 (d, J = 7.7 Hz, 4H), 6.68 (s, 1 H), 3.61 - 3.55 (m, 4H), 3.16 (hept, J = 6.9 Hz, 4H), 2.27 (p, J = 6.0 Hz, 2H), 1.30 (d, J = 6.9 Hz, 12H), 1.25 (s, 9H), 1.19 (d, J = 6.8 Hz, 12H).

[0127] 13C NMR (126 MHz, CDCI3) 6 165.0, 153.9, 144.8, 140.9, 138.3, 127.2, 123.6, 79.1 , 50.8, 28.8, 28.1 , 24.4, 23.3.

[0128] HRMS (ESI) m / z cal. for (M + H)+547.4007, found547.3968. -diisopropylphenyl)-1 ,3-diazepan-

[0129] Synthesized according to the general procedure, purified by column chromatography (diethyl ether / pentane) as a yellow solid, isolated yield 73%.

[0130] 1H NMR (500 MHz, CDCI3) 6 7.20 - 7.14 (m, 2H), 7.08 (d, J = 7.7 Hz, 4H), 6.76 (s, 1 H), 3.88 - 3.84 (m, 4H), 3.31 (m, 4H), 1.81 (p, J = 2.4 Hz, 2H), 1.32 (d, J = 6.9 Hz, 12H), 1.29 (s, 9H), 1.23 (d, J = 6.8 Hz, 12H).

[0131] 13C NMR (125 MHz, CDCI3) 6 164.1 , 157.5, 145.4, 143.3, 141.8, 126.94, 123.9, 80.1 , 56.7, 29.3, 28.2, 27.6, 24.5, 23.6.

[0132] HRMS (ESI) m / z cal. for (M + H)+561.4163, found 561.4117.

[0133] (E)-tert-butyl 2-((1 ,3-dimesityl-4,5-dimethyl-1 H-imidazol-

[0134] 2(3H)ylidene)hydrazono)acetate (EZT352) Synthesized according to the general procedure, purified by column chromatography (diethyl ether / pentane) as a yellow solid, isolated yield 78%.

[0135] 1H NMR (400 MHz, CDCI3) 56.96 (s, 1 H), 6.91 (s, 4H), 2.30 (s, 6H), 2.10 (s, 12H), 1.72 (s, 6H), 1.31 (s, 9H).

[0136] 13C NMR (50 MHz, CDCI3) 5 165.9, 150.9, 137.8, 136.2, 135.3, 132.5, 128.9, 118.1 , 79.0, 28.1 , 21.2, 18.1 , 8.8.

[0137] HRMS (ESI) m / z cal. for (M + H)+475.3068, found 475.3034.

[0138] Example 2

[0139] This example shows the cytotoxic activity of compounds of the present invention against the A2780 ovarian cancer cell line.

[0140] The compounds were tested at 2 concentrations, 10pM and 30pM, in a 48-hour 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assay. Specifically, cells were seeded in 96-well flat-bottom microplates in their standard growth medium (200 pl / well) at a density of 4-6x103cells / well. After overnight incubation, the medium was discarded and the adherent cells were treated with the mentioned concentrations of the different compounds diluted in the growth medium (200 pl / well). After 48 hours, 20 pL of MTT solution in phosphate-buffered saline (PBS) (5mg / ml) (Sigma-Aldrich) was added into each well and the plates were incubated at 37°C for 3 hours. After incubation, the content of the wells was removed carefully with 27g needle and 200pl / well dimethylsulfoxide were added to each well to dissolve the formazan crystals. Following mixing, the plates were analyzed with Multiskan™ FC Microplate Photometer (Thermo Scientific™) and the absorbance was measured at 550 nm. The untreated controls were given the arbitrary value of 100% viability / proliferation.

[0141] The results of the cytotoxic activity are shown in Table 1 below and in Figure 1.

[0142] Table 1

[0143] The results show that the compounds of the present invention suppress A2780 cell survival and proliferation in a dose-dependent manner.

[0144] Example 3 Cytotoxic capacity against A2780 cells

[0145] Compounds of the present invention were tested for cytotoxic capacity against A2780 ovarian cancer cells and their activity was compared to the activity of the widely used chemotherapeutic agent, oxaliplatin. The tests were carried out by using the protocol described in Example 2 above. The compounds were tested at concentrations 2.5pM and 20pM. The results are shown in Figure 2.

[0146] The results show that the compounds of the present invention exhibit significantly higher cytotoxicity against A2780 cells in comparison to oxaliplatin.

[0147] Cytotoxic capacity against MDA-MB-231 and HT29 cells

[0148] Compounds of the present invention were tested for cytotoxic capacity against MDA- MB-231 breast cancer cells and against HT29 colorectal adenocarcinoma cells and their activity was compared to the activity of oxaliplatin. The tests were carried out by using the MTT cell viability assay as described in Example 2. The results of 48hr exposure MDA-MB-231 cells to increasing concentrations of the tested compounds are shown in Figure 3. The corresponding results against HT29 cells are shown in Figure 4.

[0149] The results show that the compounds of the present invention exhibit significantly higher cytotoxicity against MDA-MB-231 and HT29 cells in comparison to oxaliplatin.

[0150] Example 4

[0151] The cytotoxic potency of compounds EZT230 and EZT350 was measured by using the MTT conversion assay, as described in Example 2, against eight human and two mouse cancer cell lines. The IC50 values were determined after 48-hour exposure and compared to IC50 for cisplatin and oxaliplatin, two of the most common platinum-based chemotherapeutics.

[0152] Representative dose-response curves are shown in Figure 5.

[0153] Table 2 below shows IC50 values of EZT230, EZT350, oxaliplatin and cisplatin against the tested cell lines. The IC50 values were calculated from multiple experiments (n>3), using GraphPad Prism 10.0.0 software and the AAT Bioquest, "Quest Graph™ IC50 Calculator" platform. Results are expressed as mean± SD.

[0154] Table 2

[0155] ND: Not determined.

[0156] There were little in-between differences in IC50 concentrations of EZT230 and EZT350 in each cell line tested; IC50 values ranged from approximately 0.5 pM in A2780 ovarian cancer cells (most sensitive) to 4.1 pM in HepG2 hepatocellular carcinoma cells (less sensitive) for human cancer cells or 5.6 pM in B16 melanoma mice cancer cell line. Depending on the cell line, the IC50 values for oxaliplatin and cisplatin appear to be 2 to 25-fold higher. Therefore, both EZT230 and EZT350 yielded significant cytotoxic / cytostatic effects that exceeded those of cis-platin and oxaliplatin.

[0157] Example 5

[0158] The ability of EZT230 to induce apoptosis was measured by using the Annexin V & Propidium Iodide apoptosis assay in A2780 ovarian cancer cells after 24 hours incubation with three concentrations of the compound, 1.5, 5 or 10 pM. For this purpose, the BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit I was used. Specifically, cells were seeded in 6-well flat-bottom plates in RPMI-1640 (2 ml / well) at a density of 6-7x105 cells / well. After overnight incubation, the medium was discarded and 2 ml / well fresh medium containing the drugs was added on the cells. After 24 hours, each well’s supernatant was discarded and transferred into a 15ml centrifuge tube. The wells were washed twice with PBS and the supernatant was transferred into the 15ml falcon. The cells were detached with Trypsin-EDTA and transferred into the 15ml falcon. Following centrifugation at 1 ,000 rpm for 3 minutes, the supernatant was removed, the cell pellet was resuspended in PBS, and cells were counted using a hemocytometer (Neubauer chamber) using trypan blue stain. Cells were transferred in a different 15ml falcon in PBS at a final concentration of 1x106 cells / mL. The suspension was centrifuged again at 1 ,000 rpm for 3 minutes and the supernatant was removed. The cell pellet was resuspended in 1 ml of 10-fold diluted Annexin V Binding Solution (provided by the kit). 100 pL of the abovementioned cell suspension were transferred to a round-bottom flow cytometry tube. Five pL of Annexin V, FITC Conjugate, and then 5 pL of PI Solution (both provided in the kit) were added to the cell suspension. For every sample there was an unstained control tube. The mix was incubated for 15 minutes at room temperature with protection from light. Following staining, cells were washed and resuspended in 400 pL of 10-fold diluted Annexin V Binding Solution. The analysis of fluorescence was conducted using a BD FACS Celesta™ Flow Cytometer.

[0159] The results are shown in Figures 6 and 7. In Figure 6, the Q4 squares (bottom left) in each graph capture live cells, Q3 (bottom right) show early apoptotic cells, Q1 (top left) correspond to cells that are necrotic, and Q2 late apoptotic cells (top right). Figure 7 shows the cumulative data for early and late apoptotic cells. PI: propidium iodide; Results are expressed as mean± SD and are representative of three independent experiments; ***P<0.001 ; **P<0.01 ; *P<0.05.

[0160] Compared to untreated A2780 cells, EZT230 increased apoptosis from a background death of 3.6% in control cultures to 15.3% in cells treated with 10 pM of the drug (Figure 6). The results also showed that apoptosis induction was dose-dependent with a significant increase in the percentage of early apoptosis as well as late apoptosis between 1.5 pM and 10 pM drug concentrations (Figure 7).

[0161] Example 6

[0162] The effect of EZT230 on cell cycle progression was tested to determine whether it causes cell cycle arrest, in addition to inducing cell death. To this end, A2780 ovarian cancer cells were exposed for 24 hours to two concentrations of the compound, 1.5 pM and 10 pM, before assessing the DNA content by PI staining and flow cytometry.

[0163] A2780 cells were seeded in 10cm cell culture petri dishes in RPMI-1640 (10 ml / dish) at a density of 2-2.5x106 cells / dish. After overnight incubation, the medium was discarded and the cells were treated with two different concentrations of the new compound diluted in the growth medium (10 ml / dish). After 24 hours, the supernatant was discarded and transferred into a 50ml falcon. The dishes were washed with PBS and the supernatant was transferred into the 50ml falcon. The cells were detached with Trypsin-EDTA, PBS was added and cell suspension was transferred into the 50ml falcon. The suspension was centrifuged at 1 ,500 rpm for 5 minutes and the supernatant was removed. The cell pellet was resuspended in 2 ml PBS and the suspension was centrifuged again at 1 ,500 rpm for 5 minutes. The supernatant was removed and the cell pellet was resuspended in 0.5 ml PBS. Four and a half (4,5) mis cold Ethanol 80% v / v were added dropwise to the suspension while vortexing. The suspension was transferred to a 15ml falcon and stored at 4°C overnight. The following day, the suspension was centrifuged at 1 ,500 rpm for 5 minutes and the supernatant was removed. The cell pellet was resuspended in 1 ml PBS and the suspension was centrifuged again at 1 ,500 rpm for 5 minutes. The supernatant was removed and the cell pellet was resuspended in 1 ml 0,1 % Triton X (Merck) in PBS. The mix was incubated at room temperature for 5 minutes, and then, centrifuged at 1 ,500 rpm for 5 minutes. The supernatant was removed, the cell pellet was resuspended in 1 ml PBS and incubated at room temperature for 5 minutes. The suspension was centrifuged at 1 ,500 rpm for 5 minutes and the supernatant was removed. A “Working PI staining solution” was prepared: stock PI staining buffer (Tris-HCI pH 7.5 100mM, NaCI 150mM, CaCh 1 mM, MgCh 0.5mM, NP-40 0.1 %) (4°C) + PI stock solution (1 mg / ml in ddH2O) 1 :250 + 10pl RNAase / ml from stock 10mg / ml. 100pl of working PI staining solution were added to each sample and they were incubated for 30 minutes in the dark. Then, 400pl PBS was added to each sample and the mix was transferred in round-bottom flow cytometry tube for analysis in a a BD FACS Celesta™ Flow Cytometer.

[0164] The results of the flow cytometric analysis revealed an accumulation of cells in the G0 / G1 phase in a concentration-dependent manner. (Figure 8) The percentage of cells in the G0 / G1 phase were 47.82% in the untreated cells, and 68.40 and 76.75% in the cells treated with EZT230 at 1.5 pM and 5 pM, respectively. This was paralleled by a reduction in the percentage of cells in the S and G2 / M phases. Thus, EZT230 exerts cytostatic effects by inhibit the cell cycle at G0 / G1 phase.

Claims

CLAIMS1. A compound of Formula (1)or a pharmaceutically acceptable salt thereof, wherein= = = is a single bond, or a double bond, or no bond, wherein when = = = is single bond, n is 1 , 2 or 3, when = = = is double bond, n is 1 when = = = is no bond, n is 1 ;Ri and R2 are the same or different and are selected from the group consisting of H, C1-C15 alkyl, CH2-phenyl, cycloalkyl, heteroaryl and aryl, wherein R1 and R2 are optionally substituted with one or more substituents which are the same or different and are selected from the group consisting of C1-C15 alkyl, C1-C15 alkoxy, NH2, NO2, CN, CF3, Cl, Br and I;R3and R4 are the same or different and are selected from the group consisting of C1-C15 alkyl, CH2-phenyl, cycloalkyl, heteroaryl and aryl, wherein R3 and R4 are optionally substituted with one or more substituents which are the same or different and are selected from the group consisting of C1-C15 alkyl, C1-C15 alkoxy, NH2, NO2, CN, CF3, Cl, Br and I;Rs is selected from the group consisting of H, C1-C15 alkyl, CH2-phenyl, heteroaryl and aryl, wherein Rs is optionally substituted with one or more substituents, which are the same or different and are selected from the group consisting of C1-C15 alkyl, C1-C15 alkoxy, heteroaryl or aryl, NH2, NO2, CN, CF3, F, Cl, Br and I; provided that the compound is not2. The compound according to claim 1 , or a pharmaceutically acceptable salt thereof, wherein Ri and R2 are the same or different and are selected from H, or C1-C4 alkyl.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are the same or different and are selected from a C1-C4 alkyl.

4. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein R1 is methyl and R2 is methyl.

5. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein R3 and R4 are selected from the group consisting of C1-C4 alkyl, CH2-phenyl, heteroaryl and aryl.

6. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein R3 and R4 are heteroaryl, or aryl.

7. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein R3 and R4 are mesityl, or2,6 - diisopropylphenyl.

8. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein R3 and R4 are the same and are mesityl, or 2,6 - diisopropylphenyl.

9. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein Rs is selected from the group consisting of C1-C4 alkyl, benzyl, heteroaryl and aryl.

10. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein, Rs is tert-butyl.

11. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein = = = is a double bond and n is 1.

12. The compound according to claim 1 , or a pharmaceutically acceptable salt thereof, wherein the compound is selected from(E)-benzyl 2-((1 ,3-dicyclohexyl-1 H-imidazol-2(3H)- ylidene)hydrazono)acetate;(E)-benzyl 2-((1 , 3-dimesityl- 1 H-imidazol-2(3H)-ylidene)hydrazono)acetate;(E)-benzyl 2-((bis(mesityl(methyl)amino)methylene)hydrazono)acetate;(E)-benzyl 2-((1 ,3-dimesityltetrahydropyrimidin-2(1 H)- ylidene)hydrazono)acetate; (E)-tert-butyl 2-((1 ,3-bis(2,6-diisopropylphenyl)-4,5-dimethyl-1 H-imidazol- 2(3H)-ylidene)hydrazono)acetate;(E)-benzyl 2-((1 , 3-dimesityl- 1 ,3-diazepan-2-ylidene)hydrazono)acetate;(E)-benzyl 2-((1 ,3-dimesitylimidazolidin-2-ylidene)hydrazono)acetate;(E)-ethyl 2-((1 ,3-dimesitylimidazolidin-2-ylidene)hydrazono)acetate;(E)-ethyl 2-((1 ,3-bis(2,6-diisopropylphenyl)imidazolidin-2- ylidene)hydrazono)acetate;(E)-methyl 2-((1 , 3-di mesityl- 1 H-imidazol-2(3H)-ylidene)hydrazono)acetate;(E)-tert-butyl 2-((1 ,3-bis(2,6-diisopropylphenyl)tetrahydropyrimidin-2(1 H)- ylidene)hydrazono)acetate;(E)-tert-butyl 2-((1 ,3-bis(2,6-diisopropylphenyl)-1 ,3-diazepan-2ylidene)hydrazono)acetate;(E)-tert-butyl 2-((1 ,3-dimesityl-4,5-dimethyl-1 H-imidazol-2(3H)ylidene)hydrazono)acetate.

13. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

14. The compound, or a pharmaceutically acceptable salt thereof, for use according to claim 13, wherein the cancer is selected from the group consisting of ovarian cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, liver cancer, breast cancer and melanoma.

15. A pharmaceutical composition comprising as active ingredient a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition according to claim 15, wherein the composition has the form of powder, tablet, capsule, suppository, solution, suspension, emulsion, gel, cream, ointment, spray, or transdermal patch.

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