The present invention refers to novel conjugates of the metformin molecule, and their use in the therapeutic treatment of diabetic diseases, in particular diabetic diseases associated with cardiovascular problems, and cancer diseases.

In recent decades, diabetes mellitus has assumed the characteristics and scale of a global health emergency due to its high prevalence. In 1980, the World Health Organization (WHO) estimated that around 108 million people worldwide had diabetes. This number has now quadrupled, reaching 2017 451 million diabetic patients. According to reliable projections, in 2045 the number of patients will be around 700 million.

Type 2 diabetes is the most common form among the different types of diabetes mellitus and affects 90% of cases. It develops predominantly from 40 years of age and primarily affects obese or overweight individuals. The causes of this disease can be traced back to two major pathological changes: insufficient insulin is produced to meet the body's needs (insulin secretion deficiency picture), or insulin - although produced in adequate quantities - does not act satisfactorily (insulin resistance picture). In both situations, lack of glucose control and the onset of hyperglycaemia are the most typical consequences of the disease and the most widely used diagnostic marker (American Diabetes Association. Diagnosis and Classification of Diabetes Mellitus. 2014, Diabetes Care, Vol. 37, p. S81-S90). In type 2 diabetic patients, in addition to insulin deficiency and insulin resistance, ancillary abnormalities of other hormones contributing to the control of glucose metabolism may also be observed; for example, these patients may exhibit reduced production of glucagon-like peptide 1 (GLP-1), a hormone produced by the intestine during meals which contributes to blood glucose control.

In addition to typical alterations in glucose metabolism, the diabetic state is characterized by the persistent presence of a pro-inflammatory basal condition which, among other consequences, results in damage to vascular endothelial cells and thus plays a key role in the genesis of “endothelial dysfunction”. In particular, protracted hyperglycaemia causes increased oxidative stress resulting in vascular inflammation linked to increased activity of pro-inflammatory factors, including, for example, the nuclear transcription factor NFkB. Furthermore, the presence of high plasma glucose concentrations promotes non-enzymatic glycation of proteins, with the formation of species capable, in turn, of causing damage to the endothelial wall of the vessels (Tan KC, et al., “Advanced glycation end products and endothelial dysfunction in type 2 diabetes”; Diabetes Care 2002; 25:1055-9).

Oxidative stress is one of the main pathogenetic mechanisms of dysfunction and correlates significantly with the development of a number of cardiovascular diseases (hypertension, atherosclerosis, coronary heart disease, other micro- and macro-vascular diseases, etc.), which are indeed one of the major complications associated with diabetes. Endothelial dysfunction, in particular, is a pathological condition characterized by reduced vasodilation and pro-coagulant and pro-inflammatory activity, related to a reduced production of modulatory protective factors of endothelial origin, such as nitric oxide NO (John E, et al, “Endothelial function and dysfunction”; Circulation 2007: 115:1285- 95).

Metformin, first introduced in the United States in 1995, is still the world's leading oral drug in the management of type 2 diabetes mellitus. In fact, Metformin can delay the progression of type 2 diabetes mellitus, reduce the risk of complications and mortality rates in patients by decreasing glucose synthesis (gluconeogenesis), and increase the peripheral tissues' sensitivity to insulin. Additionally, this drug improves insulin sensitivity by activating insulin receptor expression and enhancing its tyrosine kinase activity. Unlike other antidiabetic drugs commonly used in the clinic, metformin is well tolerated, has mild side effects, and has a very low risk of dangerous hypoglycaemic responses (Bailey CJ, “The Current Drug Treatment Landscape for Diabetes and Perspectives for the Future. Clinical Pharmacology & Therapeutics” 2015, Vol. 98, 2, p. 170-184).

The mechanism of action of metformin is not yet fully clarified. However, a key role in promoting the effects of the drug is the activation of the enzyme AMPK (AMP-dependent protein kinase) (Viollet B, et al, “Cellular and molecular mechanisms of metformin: an overview. Clin Sci (Lond)”, 2012, Vol. 122, 6, p. 253-270).

From a pharmacokinetic point of view, due to its chemical-physical characteristics, in particular a high level of hydrophilicity of the molecule (LogP = -1.83; a theoretical calculation using ALOGP 2.1 software), metformin has limited oral bioavailability which entails the use of considerable dosages in order to reach adequate therapeutic levels in the body (Gonga L, et al, “Metformin pathways: pharmacokinetics and pharmacodynamics”, Pharmacogenet Genomics. 2012; 22(11): 820-827.8). Indeed, absorption by passive diffusion of metformin through the different cell types is strongly disadvantaged, but rather requires the presence of monoamine transporters placed on the plasma membrane. Such absorption mechanism strongly restricts the entry of the molecule into cells and, above all, is a major obstacle in individuals with altered gene expression of these transporters (Reitman ML, and Schadt EE. “Pharmacogenetics of metformin response: a step in the path toward personalized medicine”; J Clin Invest. 2007; 117:1226-1229). Alongside this pharmacokinetic limitation, it should also be emphasized that the drug, although equipped with a pharmacodynamic mechanism capable of guaranteeing excellent effects on the metabolic aspects of diabetes disease, and in particular in glycaemic control, does not exert direct effects on cardiovascular functions, which, instead, would be a useful completion of the pharmacological profile.

In fact, due to the complexity of diabetes disease and cardiovascular complications associated therewith, the treatment of diabetic patients frequently involves the use of pharmacological “cocktails” in which properly antidiabetic drugs that aim to correct the metabolic aspects of the disease (for example, metformin itself) are associated with drugs that are instead intended to control one or more of the concomitant diseases (e.g., antihypertensive drugs, antiplatelet drugs, hypocholesterolemic drugs, etc.). Of course, the use of such “cocktails” has undoubted disadvantages in terms of poor patient compliance and increased risk of drug-drug interactions.

Metformin prodrugs are also known. Huttunen K.M., et al, Synthesis, 2008, (22): 3619-3624, describes several chemically stable metformin derivatives, potentially used as prodrugs, which include benzyl derivatives, amide derivatives, and cyclic phosphoramidates.

The studies illustrated in Markowicz-Piasecka M., et al, Oxid Med Cell Longev.; 2017, no 7303096 teach that metformin sulfonamide prodrugs, in particular cyclohexyl sulfonamide prodrug, are able to inhibit the activity of human acetylcholinesterase and butyrylcholinesterase enzymes.

Rautio J., et al, Bioorg Med Chem Let , 2014, 24(21):5034-5036 discloses a nitrogensubstituted metformin benzenesulfonamide prodrug and its in vivo activation by the enzyme glutathione-S -transferase.

Alongside the well-known antidiabetic properties, although with the above limitations, metformin is recently emerging as a possible drug for the treatment of different types of cancer (Shoeb Ikhlas, and Masood Ahmad, “Metformin: Insights into its anticancer potential with special reference to AMPK dependent and independent pathways; Life sciences 2017; 1-48).

In recent decades, anti-cancer drug therapies have made significant progress, allowing an increasing number of cases to change the once invariably poor prognosis of these diseases, making them treatable and sometimes curable. However, conventional cancer therapy is still primarily surgical intervention, if the tumor site and the non-advanced stage of the disease allow it, usually associated with chemotherapy and/or radiotherapy treatments, which are unfortunately burdened with particularly intense side effects.

In this context, therefore, there is a need for new therapeutic strategies, as alternatives to those already existing, which are effective in the treatment of diabetes diseases and/or cancer diseases and at the same time capable of beneficial protective effects on the vascular endothelium, thereby counteracting cardiovascular alterations associated with the aforementioned diseases more effectively.

In view of the above needs, one object of the invention is a compound having the formula (I): wherein,

R is:

-NH-R ¹ ,

-N=C=S, or wherein R ¹ is selected from the group consisting of: and o

— C-CH ₂ CH ₂ S-S-CH ₂ CH=CH ₂ wherein R ² is selected from

(a) linear or branched, saturated or unsaturated Ci-Cio alkyl;

(b) saturated or unsaturated C3-C12 cycloalkyl;

(c) aryl;

(d) hetero aryl;

(e) Ci-Cs alkylaryl or Ci-Cs alkyl heteroaryl; wherein each of (a), (b), (c), (d) and (e) is optionally substituted with one or more substituents selected from the group consisting of halogen, CN, NH2, NH-alkyl, NH-aryl, NH-arylalkyl, N(alkyl) ₂ , NO ₂ , OH, O-alkyl, O-aryl, O-arylalkyl, O-CO-alkyl, O-CO-aryl, O-CO-arylalkyl, CHO, CO-alkyl, CS-alkyl, CO-aryl, CS-aryl, CO-arylalkyl, CS-arylalkyl, CO-O-alkyl, CS-O-alkyl, CO-O-aryl, CS-O-aryl, CO-O-arylalkyl, CS-O-arylalkyl, CO- NH ₂ , CO-NH-alkyl, CO-NH-aryl, CO-NH-arylalkyl, CO-N(alkyl) ₂ , CF3, wherein said alkyl groups are each, independently, a linear or branched, saturated or unsaturated Ci-Cs chain.

Pharmaceutical compositions comprising a compound of formula (I) and the therapeutic use of a compound of formula (I), as defined in the attached independent claims, are further objects of the invention.

Additional features and advantages of the invention are defined in the dependent claims, which form an integral part of the specification.

The term "alkyl", as used herein, refers to a linear or branched, saturated or unsaturated hydrocarbon radical comprising one to ten carbon atoms (C1-C10), wherein the alkyl radical can optionally be independently substituted with one or more substituents as indicated above.

Exemplary C1-C10 alkyl groups include, but are not limited to, methyl, ethyl, ethylene, propyl, n-propyl, propylene, isopropyl, butyl, butylene, butadiene, isobutyl, sec-butyl, tertbutyl, pentyl, pentene, pentadiene, isopentyl, neopentyl, 1 -ethylpropyl, hexyl, hexene, hexadiene, hexatriene, isohexyl, 1,1 -dimethylbutyl, 2,2-dimethylbutyl, 3, 3 -dimethylbutyl and 2-ethylbutyl, 1 -heptyl, 1 -octyl, 1 -nonyl, 1 -decyl, and the like.

The term “cycloalkyl”, as used herein, refers to a saturated or partially unsaturated, nonaromatic monovalent ring having 3 to 12 carbon atoms (C3 ^_ Ci ₂ ). Non-limiting examples of the “C ₃ -Ci ₂ cycloalkyl group” include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo [3.2.1] octyl, adamantyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and the like.

Cycloalkyl groups are optionally independently substituted with one or more substituents as described above and can be linked to the remaining portion of the molecule of the compound of the invention through an alkyl linker as mentioned above. Within the scope of the present description, the term "aryl" indicates a monovalent aromatic hydrocarbon radical of 6 to 14 carbon atoms (C6 ^_ Ci4) derived from the removal of a hydrogen atom from a single carbon atom of a parent aromatic ring system. Representative non-limiting examples of an aryl group (C6 ^_ Ci4) include phenyl, biphenyl, 1-naphthyl, 2- naphthyl, 1-anthryl, 2-anthryl, 9-anthryl, 1 -phenanthryl, 2-phenanthryl, 3 -phenanthryl, 4- phenanthryl, 9-phenanthryl, indenyl, and the like. Aryl groups are optionally independently substituted with one or more substituents as described above and can be linked to the remaining portion of the molecule of the compound of the invention through an alkyl linker as mentioned above.

The term “heteroaryl” refers to a 5-, 6- or 7-membered monovalent cyclic aromatic radical containing one or more heteroatoms, for example 1, 2, 3, 4 or 5 heteroatoms, preferably independently selected from nitrogen, oxygen, sulfur, selenium, and tellurium. Heteroaryl groups are optionally independently substituted with one or more substituents as indicated above or fused with other aryl or heteroaryl groups as described above.

The term “alkylaryl” refers to an alkylene-(X) radical, wherein alkylene is an alkyl group as indicated above and X is an aryl or heteroaryl group as described above.

In one embodiment of the invention, R is -N=C=S.

According to another embodiment of the invention, R is -NH-R ¹ and R ¹ is preferably selected from

In this embodiment, R ² is preferably phenyl or Ci-Cs alkylphenyl.

Particularly preferred compounds within the scope of the invention are:

N-(N,N-dimethylcarbamimidoyl)carbarnimidoyl isothiocyanate having the formula (II)

(II);

N-(phenylaminothiocarbonyl)-N,N-dimethylcarbamimidoyl - carbamimidamide) having the formula (III)

NH NH S

H ₃ C A

N NH' N N

H ₃ C ^{z H H}

(ill);

N-(N-(N,N-dimethylcarbamimidoyl)carbamimidoyl phenylthioamide having the formula (IV)

(IV);

S,S-benzyl-((N,N-dimethylcarbamimidoyl)carbamimidoyl)-car bamodithioperoxoate having the formula (V)

(V).

As will be apparent from the following detailed description, the present invention provides novel compounds of formula (I) which represent “pharmacodynamic hybrids” derived from the conjugation of the native metformin drug to particular molecular moieties capable of releasing hydrogen sulfide (H2S), referred to as “H2S donors”.

Recent studies have identified hydrogen sulfide (H2S) as an endogenous “gasotransmitter”, produced ubiquitously in almost all districts of the body. Through fundamental pleiotropic effects, physiological concentrations of H2S are able to regulate the correct functioning of numerous systems, including in particular the cardiovascular system, in which the gasotransmitter promotes vasodilatory, antihypertensive and antiplatelet effects, and protective effects on the endothelial function. Physiological levels of H2S are also able to modulate the inflammatory response and activate a strong cytoprotective response predominantly linked to the activation of transcription factors of genes coding for the expression of enzymes essential in the antioxidant defence, including for example Nrf2 (Xie L, et al, “Hydrogen Sulfide Induces Keapl S- sulfhydration and Suppresses Diabetes- Accelerated Atherosclerosis via Nrf2 Activation”; 2016 Diabetes 65: 3171-84). It has also been shown that the endogenous gasotransmitter H2S, at appropriate concentrations, has inhibitory activity against the proliferation and migration of different types of cancer cells (Wu Dongdong, et al; “Hydrogen sulfide in cancer: Friend or foe?” Nitric Oxide. 2015; 50. 38-45).

The paper by Martelli A., et al, Antioxid Redox Signal. 2020; 32(2): 110- 144 discloses the use of organic isothiocyanates as H2S donors, e.g., isothiocyanates isolated from Brassicaceae plants, and their activity against oxidative stress, optionally in combination with the administration of metformin.

US 2013/0064904 discloses hydrogen sulfide prodrugs and their use for the treatment of various diseases, including cardiovascular diseases, also in combination therapies, for example in combination with biguanide drugs.

The present invention is based on the results obtained by the inventors in the experimentation and research activities described in the following experimental section. In short, in vitro studies carried out by the present inventors have shown that the compounds of the invention are capable of simultaneously releasing the metformin molecule and hydrogen sulfide, thus representing prodrugs, and that this release is favoured under experimental conditions mimicking the intracellular environment, such as for example the presence of a high concentration of thiols. Further studies have also confirmed that the compounds of the invention retain the ability of metformin to induce glucose uptake by human liver cells and showed that the activity exerted by the compounds of the invention is surprisingly superior compared to the metformin drug alone. By further examining the in vitro activity of the compounds of the invention, the present inventors have also found that these compounds are advantageously able to promote the cellular release of the intestinal peptide hormone GPL- 1, known for its blood glucose control. In accordance with the results of the in vitro experiments, the marked hypoglycaemic activity of the compounds of the invention was subsequently also validated in vivo by using an animal model of diabetic transgenic mice.

Following treatment of different cancer cells with the compounds of the invention, including pancreatic or mammary adenocarcinoma metastatic cells, the present inventors also detected a concentration-dependent H2S intracellular release and noted a surprising reduction in viability and proliferative capacity of cancer cells, which is significantly higher than the same effects achieved with metformin alone.

In the light of the above, the compounds according to the invention therefore represent an important innovative therapeutic tool, particularly in the field of diabetic and cancer diseases. As a result of the biotransformation reactions occurring within the human body, the compounds of the invention in fact lead to the formation of metformin and the simultaneous release of hydrogen sulfide, providing a surprising synergistic action between the euglycemic and/or anticancer effects of the metformin drug and the beneficial cardiovascular, endothelium-protective and/or anticancer effects of the H2S gas. In addition to these important pharmacodynamic advantages, the compounds forming the object of the present invention also act as metformin prodrugs by reducing the polarity of this molecule and thus allowing it to cross the plasma cell membrane more effectively, independently of the monoamine transporters.

Thanks to the beneficial activities on glucose metabolism and on the reduction of the viability and proliferation of cancer cells, the compounds according to the invention are therefore suitable for therapeutic use, particularly for the treatment of diabetic diseases, cardiovascular diseases and/or cancer diseases.

The compounds according to the invention can be conveniently used in the therapeutic treatment of diabetic disease, in particular type 2 diabetes mellitus, and other alterations in glucose metabolism requiring the use of metformin, as well as in the treatment of cardiovascular diseases, in particular diabetes-related cardiovascular diseases, such as for example hypertension, microvascular complications (retinopathy, neuropathy, nephropathy), macrovascular complications (coronary heart disease, cerebrovascular disease, peripheral vascular disease), and erectile dysfunction.

Cancer diseases that can be treated with the compounds according to the invention include, but are not limited to, pancreatic cancer, breast cancer, liver cancer, lung cancer, melanoma and other types of neoplastic diseases.

A pharmaceutical composition comprising a compound of the invention as defined above, in combination with at least one pharmaceutically acceptable carrier, excipient and/or diluent, is also within the scope of the invention.

According to the invention, the pharmaceutical composition is suitable for use in the above therapeutic medical applications relating to the compound according to the invention.

The pharmaceutical composition of the present invention can be formulated into any suitable dosage form, for example for administration via the enteral (oral or gastro-enteral, rectal, sublingual), parenteral (intravenous, intraarterial, transcutaneous, intramuscular, intradermal, subcutaneous, intraperitoneal), topical (direct contact of the drug with the site of action and/or the skin and/or the mucous membranes), and inhalation routes.

Preferred oral dosage forms are tablets, capsules, sachets, powders, granules, pellets, gels, syrups, elixirs, oral solutions, suspensions or emulsions. The most preferred are tablets and capsules.

Of course, the selection of suitable carriers, excipients and/or diluents is carried out depending on the desired form of administration and this selection is within the skills of those of ordinary skill in the art. The selection of the dose of active principle and the dosage regimen also falls within the skills of those of ordinary skill in the art, and the selection thereof depends on several factors, such as for example the age and weight of the patient, as well as the degree of progression of the disease.

A further object of the present invention is a method for the therapeutic treatment of a diabetic disease, a cardiovascular disease and/or a tumor disease in a subject in need thereof, which comprises administering a compound as defined above or a pharmaceutical composition as defined above to said subject.

Yet another object of the present invention is a method for reducing the blood glucose level in a subject in need thereof, which comprises administering a compound as defined above or a pharmaceutical composition as defined above to said subject.

The experimental section that follows is provided for illustration purposes only and does not limit the scope of the invention as defined in the appended claims. In the experimental section, reference is made to the accompanying drawings, wherein:

Figure 1 illustrates graphs showing the formation of hydrogen sulfide (H2S) over time, after incubation of the compounds of the invention (compounds of formulas II, III and IV) in aqueous solution, in the presence or absence of L-cysteine (Cys). In particular, graphs 1A and 1C show that the compounds II and IV only release H2S in the presence of organic thiols represented by L-cysteine excess (4 mM Cys), whereas the release is virtually absent in the absence of organic thiols. This profile therefore depicts the compounds of the invention II and IV as “smart” H2S donors capable of releasing H2S not spontaneously, but only in the presence of significant concentrations of organic thiols (e.g., in the cell cytosol); graph IB instead shows that the compound III releases H2S both in the presence and in the absence of organic thiols; this profile is useful where the release of H2S is required under low presence of reduced thiols (e.g., in the case of tissues subjected to high oxidative stress);

Figure 2 shows (A) a chromatogram relating to the compound II of the invention, analysed 30 minutes after its dissolution in aqueous solution and (B) a chromatogram relating to the compound II of the invention enriched with metformin, analysed 30 minutes after its dissolution in aqueous solution;

Figure 3 shows histograms representing glucose levels in the extracellular medium of HepG2 cell cultures. A) Glucose levels in the extracellular medium in the absence of HepG2 cells. (B) Glucose levels in the extracellular medium in the presence of HepG2 cells. The reduction in the levels of this molecule in the extracellular medium indicates that the cells have taken up glucose. (C) Glucose levels in the extracellular medium in the presence of HepG2 cells, following incubation with the compound of the invention (compound of formula II) at increasing concentrations (10 |iM, 50 |iM, 100 |aM). All tested concentrations show an increase in the ability of the cells to uptake glucose. (D) Glucose levels in the extracellular medium in the presence of HepG2 cells, following incubation with metformin at increasing concentrations (10 pM - 5 mM). An increase in the ability of the cells to uptake glucose is only observed at the highest drug concentrations;

Figure 4 shows histograms representing the amount of GLP-1 hormone released by STC-1 enteroendocrine cells after the following treatments: the compound of formula II of the invention at a concentration of 100 pM, metformin at a concentration of 100 pM, or the compound JT010 at a concentration of 1 pM (a known activator of TRPA1 channels, used as a reference drug capable of promoting GLP-1 release). The statistical analysis (one-way ANOVA followed by Bonferroni’s post-hoc test) was performed by comparing the different treatments with the vehicle. ** denotes P<0.01; *** denotes P<0.001;

Figure 5 depicts a graph (A) showing blood glucose levels (expressed as the difference from the baseline value at time 0) measured at 30, 60, 90 and 120 minutes in db/db strain transgenic diabetic mice, following oral administration of the vehicle, 300 mg/kg of weight of metformin, or 300 mg/kg of weight of the compound of formula II of the invention. Graph (B) shows the relevant areas under the curve for the curves in graph (A);

Figure 6 depicts two graphs showing the viability of AsPc-1 cells (pancreatic adenocarcinoma), expressed as % of the vehicle, after treatment for 72 hours (A) with metformin (Met) and (B) with the compound of the invention (the compound of formula II; Compl) at the indicated concentrations. The statistical analysis (one-way ANOVA followed by Bonferroni’s post-hoc test) was performed by comparing the different treatments with the vehicle. ** denotes P<0.01; *** denotes P<0.001;

Figure 7 depicts two graphs showing the viability of MIAPaCa-2 cells (pancreatic adenocarcinoma), expressed as % of the vehicle, after treatment for 72 hours (A) with metformin (Met) and (B) with the compound of the invention (the compound of formula II; Compl) at the indicated concentrations. The statistical analysis (one-way ANOVA followed by Bonferroni’s post-hoc test) was performed by comparing the different treatments with the vehicle. ** denotes P<0.01; *** denotes P<0.001;

Figure 8 depicts two graphs showing the viability of MCF-7 cells (breast adenocarcinoma), expressed as % of the vehicle, after treatment for 72 hours (A) with metformin (Met) and (B) with the compound of the invention (the compound of formula II; Compl) at the indicated concentrations. The statistical analysis (one-way ANOVA followed by Bonferroni’s post-hoc test) was performed by comparing the different treatments with the vehicle. *** denotes P<0.001;

Figure 9 depicts two graphs showing the viability of MCF-10A cells (non-cancerous breast epithelial cells), expressed as % of the vehicle, after treatment for 72 hours (A) with metformin (Met) and (B) with the compound of the invention (the compound of formula II; Compl) at the indicated concentrations. The statistical analysis (one-way ANOVA followed by Bonferroni’s post-hoc test) was performed by comparing the different treatments with the vehicle. *** denotes P<0.001;

Figure 10 shows the intracellular release of hydrogen sulfide (FPS) by the compound of the invention (the compound of formula II) in AsPc-1 cells (pancreatic adenocarcinoma). Graph (A) on the left illustrates the intracellular release of H2S expressed as the fluorescence index (FI) as a function of time, following incubation with the indicated treatments. Graph (B) on the right shows the area under the curve resulting from the analysis of the H2S release kinetics, expressed as the fluorescence index (FI). The statistical analysis (one-way ANOVA followed by Bonferroni’s post-hoc test) was performed by comparing the different treatments with the vehicle. *** denotes P<0.001;

Figure 11 shows the intracellular release of hydrogen sulfide (H2S) by the compound of the invention (the compound of formula II) in MIAPaCa-2 cells (pancreatic adenocarcinoma). Graph (A) on the left illustrates the intracellular release of H2S expressed as the fluorescence index (FI) as a function of time, following incubation with the indicated treatments. Graph (B) on the right shows the area under the curve resulting from the analysis of the H2S release kinetics, expressed as the fluorescence index (FI). The statistical analysis (one-way ANOVA followed by Bonferroni’s post-hoc test) was performed by comparing the different treatments with the vehicle. *** denotes P<0.001;

Figure 12 shows the intracellular release of hydrogen sulfide (H2S) by the compound of the invention (the compound of formula II) in MCF-7 cells (breast adenocarcinoma). Graph (A) on the left illustrates the intracellular release of H2S expressed as the fluorescence index (FI) as a function of time, following incubation with the indicated treatments. Graph (B) on the right shows the area under the curve resulting from the analysis of the H2S release kinetics, expressed as the fluorescence index (FI). The statistical analysis (one-way ANOVA followed by Bonferroni’s post-hoc test) was performed by comparing the different treatments with the vehicle. *** denotes P<0.001;

Figure 13 shows the intracellular release of hydrogen sulfide (H2S) by the compound of the invention (the compound of formula II) in MCF-10A cells (non-cancerous breast epithelial cells). Graph (A) on the left illustrates the intracellular release of H2S expressed as the fluorescence index (FI) as a function of time, following incubation with the indicated treatments. Graph (B) on the right shows the area under the curve resulting from the analysis of the H2S release kinetics, expressed as the fluorescence index (FI). The statistical analysis (one-way ANOVA followed by Bonferroni’s post-hoc test) was performed by comparing the different treatments with the vehicle;

Figure 14 depicts a graph showing the viability of MCF-7 (breast adenocarcinoma) and MCF-10A cells (non-cancerous breast epithelial cells), expressed as % of the vehicle, after treatment for 72 hours with the compound of the invention (the compound of formula II; Compl) at the indicated concentrations. The statistical analysis (two-way ANOVA) was performed by comparing the two curves. *** denotes P<0.001;

Figure 15 depicts, on the left, a histogram showing the percentage of cells in the different phases of the cell cycle following treatment for 72 hours with the vehicle or the compound of the invention (the compound of formula II; Compl) at lOpM, 20pM and lOOpM concentrations. The statistical analysis (one-way ANOVA followed by Bonferroni’s post-hoc test) was performed by comparing the different treatments with the vehicle. ** denotes P<0.01; *** denotes P<0.001. On the right, the graphs illustrate the distribution of the cells in the different phases of the cell cycle following treatment for 72 hours with the vehicle or with lOpM, 20pM, and lOOpM Compl;

Figure 16 depicts, on the left, a histogram showing the percentage of cells in the early apoptosis phase (measured as the expression of annexin V) following treatment for 72 hours with the vehicle or the compound of the invention (the compound of formula II; Compl) at lOpM, 20pM and lOOpM concentrations. The statistical analysis (one-way ANOVA followed by Bonferroni’s post-hoc test) was performed by comparing the different treatments with the vehicle. * denotes P<0.05; ** denotes P<0.01. On the right, the graphs illustrate the distribution of the cells in the apoptosis phases following treatment for 72 hours with the vehicle or with 20pM Compl;

Figure 17 depicts, on the left, a histogram showing the percentage of cells in the early apoptosis phase (measured as the activity of the caspase 3/7 enzymes) following treatment for 72 hours with the vehicle or the compound of the invention (the compound of formula II; Compl) at lOpM, 20pM and lOOpM concentrations. The statistical analysis (one-way ANOVA followed by Bonferroni’s post-hoc test) was performed by comparing the different treatments with the vehicle. ** denotes P<0.01; *** denotes P<0.001. On the right, the graphs illustrate the distribution of the cells in the apoptosis phases following treatment for 72 hours with the vehicle or with 20pM Compl.

EXAMPLES

Example 1: The method of synthesis of the compounds according to the invention

The preparation of the compounds according to the invention is described in the following specific examples.

Compound of formula II

The method set up by the present inventors for synthesising the compound of formula II according to the invention is set out in the diagram below: i: IM NaOH, 30 minutes, r.t. ii: NEt3, CS2, tosyl chloride, IN HC1, THF.

In particular, 1.0 g (6.0 mmol) of metformin hydrochloride (N,N- dimethyldiguanidine hydrochloride) was solubilized in 10 ml of IM NaOH and the resulting mixture was stirred for 30 minutes at room temperature. The water was then evaporated under reduced pressure and the residue was added with 30 ml of methanol. The solvent was evaporated again, and the resulting residue was taken up in 20 ml methanol. The inorganic salts were removed by filtration under reduced pressure and the filtrate evaporated to dryness provided the free base metformin (0.772 g, yield = 99%). Triethylamine (NEts, 0.75 ml, 5.4 mmol) was added dropwise at 0 °C to a metformin solution (0.160 g, 1.20 mmol) in 1.0 ml THF. Then 0.16 ml (2.6 mmol) of CS2 was added dropwise. The mixture was stirred at 0 °C for 15 minutes and then left at room temperature for one hour. Tosyl chloride (0.297 g, 1.56 mmol) in 1.0 ml of THF at 0°C was then added and the mixture was stirred at room temperature for one hour. Then 1.5 ml of IN HC1 was added. The reaction mixture was then spiked with diethyl ether (EtiC); 2.0 ml) and stirred for 5 minutes at room temperature. The compound was then extracted with Et20, and the organic layer was dried over MgSCE, filtered, and finally evaporated under reduced pressure. The raw compound of formula (II) was thus obtained and purified by silica gel flash chromatography (ethyl acetate (AcOEt):petroleum ether 40-60°C = 7:3). Yield: 25%. Melting point = 225 °C. ' H-NMR (400 Hz, DMSO-d6): 8 6.97 (3H, bs), 3.01 (6H, s) ppm. ¹³ C-NMR (100 Hz, DMSO-d6): 8 176.53, 166.02, 164.36, 36.01 ppm. HRMS (ESI) m/z Calcd for C5H10N5S+: 172.06514; Found: 172.06525 [M+H]+.

Compound of formula III

The synthesis of the compound of formula III was carried out by following the method below: i: phenylisothiocyanate, DIPEA, anhydrous DMF, overnight, 80 °C.

0.15 ml (1.27 mmol) of phenylisothiocyanate and 0.33 ml (1.91 mmol) of diisopropylamine (DIPEA) were added dropwise at 0°C to a solution of 0.200 g (1.21 mmol) of metformin hydrochloride in 1.0 ml of anhydrous DMF. The resulting mixture was stirred overnight at 80°C. Then 5.0 ml of IN HC1 was added. The compound was then extracted with ethyl acetate, and the organic layer was dried over MgSCE, filtered, and finally evaporated under reduced pressure. The raw compound of formula III was thus obtained and purified by silica gel flash chromatography (ethyl acetate (AcOEt):petroleum ether 40-60°C = 2:8). Yield: 25%. Melting point = 207-209°C. ’H-NMR (400 Hz, DMSO-d6): 8 8.87 (1H, s), 7.76 (2H, d, J = 7.6 Hz), 7.24-7.20 (2H, m), 6.89 (1H, t, J = 7.2 Hz), 6.35 (2H, bs), 3.06 (6H, s) ppm. ¹³ C-NMR (100 Hz, DMSO-d6): 8 167.20, 166.14, 164.69, 141.12, 128.72, 121.59, 119.84, 36.20 ppm. HRMS (ESI) m/z Calcd for CnHi ₅ N ₆ ⁺ : 231.13527; Found: 231.13492 [M+H] ⁺ .

Compound of formula IV The compound of formula IV was synthesized by the inventors using the following method: i: Lawesson's reagent, xylene, overnight, reflux, ii: NaH, tetrahydrofuran: dimethylformamide (1:1), overnight, 50°C.

2.377 g (5.88 mmol) of Lawesson's reagent was added to a solution of 0.92 ml (7.34 mmol) of methyl benzoate in 22 ml of xylene. The resulting mixture was heated to reflux overnight. After cooling, the solvent was allowed to evaporate under reduced pressure and the raw product obtained (O-methylbenzothioate) was purified by silica gel flash chromatography (petroleum ether 40-60°C). 0.070 g of NaH (1.82 mmol, 60% mineral dispersion) was then added at 0°C to a solution of 0.200 g (1.21 mmol) of metformin hydrochloride in 3.0 ml of tetrahydrofuran: dimethylformamide (2:1). After about 15 minutes, 0.255 g (1.67 mmol) of O-methylbenzothioate was added. The resulting mixture was heated to 50°C overnight. After cooling, 10 ml of water was added to the mixture, the compound was then extracted with ethyl acetate, and the organic layer was dried over MgSO4, filtered, and finally evaporated under reduced pressure. The raw compound of formula IV was thus obtained and purified by silica gel flash chromatography (ethyl acetate:petroleum ether 40-60°C = 1:9).

Yield: 30%. Melting point = 155-157°C. 'H-NMR (400 Hz, DMSO-d6): 8 8.32- 8.29 (2H, m), 7.53-7.45 (3H, m), 6.84 (2H, bs), 3.21 (3H, s), 3.11 (3H, s) ppm. ¹³ C-NMR (100 Hz, DMSO-d6): 8 169.83, 167.56, 166.02, 137.64, 131.54, 128.58, 128.22, 36.16 ppm. HRMS (ESI) m/z Calcd for CnHi ₄ N ₅ ⁺ : 216.12437; Found: 216.12437 [M+H] ⁺ .

O-methylbenzothioate: ’H-NMR (400 Hz, DMSO-d6): 8 8.15-8.12 (2H, m), 7.67-7.64 (1H, m), 7.52-7.48 (2H, m), 4.29 (3H, s) ppm (Huang YQ, et al., “Synthesis of 5-(l- Alkoxyalkylidene)tetronates by Direct Condensation Reactions of Tetronates with Thionolactones and Thionoesters”; J. Org. Chem. 2021, 86, 2359-2368). The purity of the compounds of the invention of formulas II, III and IV thus obtained was determined by HPLC analysis using a Shimadzu LC-20AD SP apparatus with a DDA detector at 254 nm (Cl 8 column (250 mm x 4.6 mm, 5 pm, Shim-pack)). The mobile phase, delivered at isocratic flow, consisted of acetonitrile (80-50%) and water (20-50%) and a flow rate of 1.0 ml/min. The compounds were >95% pure.

The melting points were determined with a Reichert Kofler apparatus and were uncorrected. Routine nuclear magnetic resonance spectra were recorded in DMSO-d6 solution with a Bruker AVANCE apparatus ( ^J H, 400 MHz, ¹³ C, 100 MHz). Silica gel 60 (230-400 mesh) was used for column chromatography.

ESI mass spectra were acquired with two different high resolution mass spectrometers: TripleTOF® 5600+ (AB Sciex, Framingham, MA, USA), Thermo LTQ Orbitrap XL (Thermo Fisher Scientific, San Jose, CA, USA), both operating with an ESI probe. All spectra were acquired by direct infusion into the spectrometer (positive ion mode).

The chemical reactions described can be readily modified to prepare the compounds of this invention. For example, the compounds according to the invention can be successfully synthesized by making changes apparent to those skilled in the art, such as appropriately protecting interfering groups, using other suitable reagents known in the art other than those described, and/or making routine changes to reaction conditions. Alternatively, other reactions described herein or known in the art will be recognized as applicable to prepare other compounds of the invention.

Example 2: Assessment of hydrogen sulfide (H2S) release by the compound of the invention

The assessment of the release of hydrogen sulfide (H2S) by the compounds II, III and IV of the invention was carried out by the inventors in vitro, in an environment free of biological substrates, through an Apollo4000 Free Radical Analyser (WPI) connected to HiS-scnsitivc electrodes. Before incubating the compounds of the invention, the electrode was immersed in phosphate buffer pH 7.4 in the presence or absence of 4 mM L-cysteine for 20 minutes to stabilize the electrical signal. Subsequently, the compounds of the invention (the compounds of formulas II, III and IV) were incubated in PBS at a final concentration of 1 mM with a DMSO percentage of 1%, and the release of H2S, measured as the current change in pA, was monitored for 30 minutes. In order to track down the concentration of H2S released, the pA change was compared with that obtained after incubation of 1 pM NaHS in PBS at pH 4.0.

This study showed that, in the presence of organic thiols, represented by L-cysteine excess, the compounds of formulas II, III and IV have an H2S donor profile capable of releasing H2S slowly and gradually. Furthermore, as shown in Figures 1A and 1C, the compounds II and IV of the invention release H2S in a “smart” manner, that is, only in the presence of organic thiols that serve to mimic a biological environment. Graph IB shows that the compound III releases H2S both in the presence and absence of organic thiols, indicating that this compound may instead be more useful if the release of H2S is required in conditions of relative deficiency of reduced thiols, for example in tissues subjected to oxidative stress.

Example 3: Assessment of metformin release by the compound of the invention

The present inventors subsequently assessed, by HPLC analysis, whether the release of H2S described in the preceding paragraph is accompanied by the simultaneous formation of metformin, i.e., the ability of the compound of the invention to act as a metformin H2S -donor prodrug.

The release of metformin by the compound of formula II of the invention was tested in vitro by HPLC analysis using a Shimadzu LC-20AD SP apparatus with a DDA detector at 254 nm (Cl 8 column (250 mm x 4.6 mm, 5 pm, Shim-pack)). The mobile phase, delivered at isocratic flow, consisted of acetonitrile and a 10 mM ammonium acetate buffer solution (pH 5.0) in a ratio of 90:10 (v/v) at a flow rate of 1.0 ml/min at room temperature.

In particular, a sample containing the compound of the invention (compound of formula II, 1 mM), dissolved in phosphate buffer pH 7.4 in the presence of 4 mM L-cysteine, was diluted in methanol up to a concentration of 0.05 mM (SOLUTION 1) and analysed by HPLC 30 minutes after sample preparation. The chromatogram (Figure 2A) revealed the presence of two peaks: one related to the compound of the invention (retention time = 2.73 minutes, 93.5%) and one with a retention time of 11.0 minutes (6.5 %). The analysis was then repeated on a metformin-enriched sample obtained by mixing solution 1 with an equal volume of a solution containing 1 mg/ml of metformin in methanol: the chromatogram (Figure 2B) still exhibited the same two peaks, as well as an increase in the peak area with the retention time of 11 minutes, which was thus attributed to metformin.

The evidence obtained by the inventors therefore indicates that, under the conditions described above, metformin is indeed released and the compound of formula II of the invention can represent a prodrug capable of releasing metformin together with H2S. It is also apparent to the person skilled in the art that, in addition to the release of H2S by the compounds of formulas III and IV, too, metformin release may occur following hydrolytic reactions and therefore the compounds of formulas III and IV may also represent prodrugs capable of releasing metformin together with H2S.

Example 4: In vitro hypoglycaemic effects of the compounds of the invention

The present inventors studied the effects of the compounds according to the invention on glucose metabolism using a known in vitro model, and the data obtained were compared with the effects produced by metformin. In particular, test compounds were assessed for their ability to increase glucose uptake by human liver cells (HepG2 cell line).

HepG2 cells were plated (20,000 cells per well) in transparent 96-well plates. After 24 hours, the plating medium was replaced with low-glucose fresh medium (1000 mg/1) to allow the cells to adhere to the bottom of the well. After 24 hours, the medium was replaced with 100 pl of high-glucose medium (4,500 mg/1) and the cells were treated for 24 hours with the vehicle (1% DMSO), with the compound of the invention (compound of formula II) at the following concentrations: 100 nM, 500 nM, 1 pM, 5 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 mM and 5 mM, or with metformin at concentrations of 100 pM, 500 pM, 1 mM and 5 mM.

At the end of the treatment, the probe WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5- tetrazolium]- 1,3 -benzene disulfonate) (Roche, Basel, Switzerland) was added in a ratio of 1:10 relative to the volume of each well, for cell viability assessment. After 60 minutes at 37°C, the absorbance was measured at a wavelength of 450nM using a spectrophotometer (EnSpire, Perkin Elmer).

The glucose in the culture medium after the treatments was measured using the Glucose Colorimetric Detection Kit (Thermo Fisher). Briefly, 10 pl of the supernatant were diluted in 390 pl of buffer. 20 pl of the diluted samples were incubated in the dedicated plates provided in the kit with 25 pl of IX Horseradish Peroxidase (HRP), 25 pl of substrate, and 25 pl of IX glucose oxidase. After 30 minutes at room temperature, the absorbance was measured at a wavelength of 450nM using a spectrophotometer (EnSpire, Perkin Elmer). Data were analysed by GraphPad Prism 6 using a curve fitting the calibration curve obtained by incubating known concentrations of glucose (32 mg/ml, 16 mg/ml, 8 mg/ml, 4 mg/ml, 2 mg/ml, 1 mg/ml).

As shown in Figure 3, the experiments carried out demonstrated the ability of the compound according to the invention to increase the ability of Hep-G2 liver cells to uptake glucose, thus reducing the concentration of glucose in the culture medium. Surprisingly, these effects were achieved at concentrations of the compound of the invention at least 10 times lower than those at which the reference metformin drug promoted equivalent effects.

Example 5: Assessment of the activity of the compounds of the invention on the release of the GLP-1 hormone

In order to provide further validation of the hypoglycaemic activity of the compounds according to the invention, the present inventors studied the effects of these compounds on the release of the GLP-1 hormone using a known in vitro model with the STC-1 cell line (murine intestinal neuroendocrine carcinoma cells), and the data obtained were compared with the effects produced by metformin and by a TRPA1 channel activator (JT010). STC-1 cells were plated (1.5 x 10 ⁶ cells per well) in transparent 6-well plates. After 48 hours, in order to allow the cells to adhere to the bottom of the well and reach confluency, the plating medium was aspirated, and the cells were washed 3 times with low-glucose Hank's Balanced Salt Solution (1000 mg/1). Then, 1 ml of Hank’s Balanced Salt Solution containing 20 mM HEPES, pH 7.4, was added over 2 hours. At the end of the 2 hours, the solution was aspirated and replaced with 900 pl of Hank’s Balanced Salt Solution containing 20 mM HEPES, pH 7.4 and with one of the following substances: 100 pl of the compound of formula II of the invention, metformin at a final concentration of 100 pM, or JT010 compound at a concentration of IpM, with a final DMSO percentage of 1%. After 30 minutes, the supernatant was aspirated and stored at -20°C. A Merck Elisa kit (cat. EZGLP1T-36K) was used to measure total GLP-1. The samples were thawed at the time of the experiment and the kit instructions were followed. The GLP-1 released by the cells was quantified by interpolating the data with the calibration line.

As shown in Figure 4, the compound according to the invention (compound of formula II) is able to promote a significantly higher cell release of the GLP-1 hormone compared to metformin and the JT010 compound, thereby confirming the surprising hypoglycaemic activity of the compound of the invention and its superiority over metformin alone. The increase in the levels of GLP-1 (a hormone known to contribute to blood glucose control) or the trigger of “GLP-1 mimetic” mechanisms, in fact, are considered to be interesting mechanisms of action of innovative antidiabetic drugs belonging to the incretin family. However, these mechanisms are not part of the antidiabetic mechanisms of metformin, as is apparent from Figure 4, which shows that metformin does not promote any effect on GLP- 1 release. Instead, the compound II of the invention is surprisingly capable of stimulating the release of this hormone, thus showing a further important additional antidiabetic mechanism, not present in metformin, which is extremely beneficial in the clinical use of the compound of formula II.

Example 6: In vivo assessment of the activity of the compounds of the invention on blood glucose in db/db diabetic mice by oral glucose tolerance test In order to assess the effect of the compounds of the invention on the glycaemic parameters in vivo, transgenic diabetic mice (db/db) were subjected to an oral glucose tolerance test.

Specifically, 21 18-week-old male mice (mean fasting blood glucose value: 305.4 ± 17.9 mg/dL) were fasted 16 hours prior to the experiment, giving them ad libitum access to water. Then, the animals were weighed, randomized into groups of 7 each and subjected to the following gavage treatments: reference drug (metformin 300 mg/kg of weight), compound of formula II of the invention (300 mg/kg of weight) or vehicle (0.5% aqueous methyl cellulose solution).

Thirty minutes after the start of the treatments as described above, a blood sample was taken from each animal by making a small cut in the tail, and the basal venous blood glucose was measured at “time 0” (in mg/dl) using a glucometer (Glucocard G meter, Menarini diagnostics®). Subsequently, the glucose load curve was performed by administering an aqueous glucose solution (2 g/kg) to each animal through gavage. Venous blood glucose was then measured at regular time intervals (particularly 30, 60, 90 and 120 minutes after glucose gavage) by taking a venous blood sample from the tail. The values were reported as the difference from the blood glucose level observed at time 0. The respective areas under the curve (expressed as mg/dl x min) were also obtained from the curves.

The results of the in vivo study carried out by the inventors, shown in Figure 5, demonstrated that the compound of the invention surprisingly shows greater efficacy than metformin in restoring normal blood glucose levels in db/db diabetic mice subjected to oral glucose tolerance test, as well as a higher rate in producing this effect, evidenced by the fact that already 30 minutes after the glycaemic peak the blood glucose had been reduced by more than 65% of its initial value by the compound of formula II, whereas with metformin, after the same time, this reduction was only about 30%.

Example 7: Anticancer activity of the compounds of the invention

During the experimental activities, the inventors examined the H2S donor properties of the compound 1 of the invention as well as the anticancer effects of this molecule on several cell models of different types of cancer and in non-cancerous reference models. In particular, the compound of the invention (compound of formula II) was assayed on human cells of: pancreatic adenocarcinoma (AsPc-1 and MIAPaCa-2), breast adenocarcinoma (MCF-7), and non-cancerous breast epithelium (MCF-10A).

AsPC-1 cells (human pancreatic adenocarcinoma metastasis cell line) were grown in RPMI- 1640 medium (Sigma Aldrich) containing 10% fetal bovine serum (FBS - Sigma Aldrich), L-Glutamine (Sigma Aldrich) at a final concentration of 2 mM, sodium pyruvate (Sigma Aldrich) at a final concentration of 1 mM, and 1% mixture of penicillin (100 units/ml) and streptomycin (100 pg/ml) (Sigma Aldrich).

MCF-7 cells (human breast adenocarcinoma pleural metastasis cell line) and Hep G2 cells (human hepatocarcinoma cell line) were grown in EMEM medium (Sigma Aldrich) containing 10% FBS (Sigma Aldrich), L-Glutamine (Sigma Aldrich) at a final concentration of 2 mM, 1% non-essential amino acids (Sigma Aldrich), and 1% mixture of penicillin (100 units/ml) and streptomycin (100 pg/ml) (Sigma Aldrich).

MIAPaCa-2 cells (human pancreatic adenocarcinoma metastasis cell line) were grown in DMEM medium (Sigma Aldrich) containing 10% FBS (Sigma Aldrich), L-Glutamine (Sigma Aldrich) at a final concentration of 2 mM, and 1% mixture of penicillin (100 units/ml) and streptomycin (100 pg/ml) (Sigma Aldrich).

MCF-10A cells (human breast epithelial non-tumor cell line) were grown in the growth medium designated as Mammary Epithelial Cell Growth Medium Bullet Kit (EuroClone) spiked with cholera toxin at a final concentration of 100 ng/ml.

All cell lines were grown in T75 cell culture flasks in an incubator at 37°C with 5% CO2, up to 80% confluence.

Cell viability assessment

Once the confluence was reached, 10 ³ cells per well were plated in transparent 96- well plates. After 24 hours, in order to allow the cells to adhere to the bottom of the well, the plating medium was replaced and the cells were treated for 72 hours with the vehicle (1% DMSO), the compound of the invention (compound of formula II) at concentrations of 100 nM, 500 nM, 1 pM, 5 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 mM and 5 mM, or metformin (Met) at concentrations of 100 pM, 500 pM, 1 mM and 5 mM. At the end of the treatment, the probe WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolium]-l,3 -benzene disulfonate) (Roche, Basel, Switzerland) was added in a ratio of 1:10 relative to the volume of each well, for cell viability assessment. After 60 minutes at 37°C, the absorbance was measured at a wavelength of 450 nM using a spectrophotometer (EnSpire, Perkin Elmer).

As shown in Figure 6, treatment of AsPc-1 cells (pancreatic adenocarcinoma) with the metformin molecule (Met) had no effect on cell viability. Instead, the compound of the invention induced a significant, concentration-dependent reduction in cell viability, with an IC50 of 1.745 x 10' ⁴ M and a maximum efficacy of approximately 95% at a concentration of 1 mM.

Treatment of MIAPaCa-2 cells (pancreatic adenocarcinoma) with metformin (Met; Figure 7) caused a modest effect on cell viability only at the highest concentrations used (5mM and lOmM), whereas the compound of the invention induced a concentration-dependent reduction in cell viability, with an IC50 of 3.586 x 10' ⁴ M and a maximum efficacy of approximately 95% at a concentration of 10 mM.

Even on MCF-7 cells (breast adenocarcinoma), as shown in Figure 8, treatment with metformin caused a modest effect only at the 5 mM concentration. Instead, treatment with the compound of the invention resulted in a concentration-dependent reduction of cell viability, with an IC50 of 1.9 x 10' ⁵ M and a complete reduction of cell viability at a concentration of 5 mM.

Treatment of MCF-10A cells (non-cancerous breast epithelial cells) with metformin led to a significant decrease in viability at the 1 mM and 5 mM concentrations (Figure 9), whereas the compound of the invention caused an all-or-nothing viability reduction with an IC50 of 1.72 x 10' ⁴ M and a maximum efficacy of approximately 90% at the 5 mM concentration. Example 8: Assessment of hydrogen sulfide release within cancer cells

In order to assess the release of hydrogen sulfide (H2S) within AsPc-1, MIAPaCa-2, MCF- 7 and MCF-10A cells, the present inventors carried out a spectrofluorimetric analysis. The cells were plated into black 96-well plates at a density of 72xl0 ³ cells per well and after 24 hours incubated with a solution of WSP-1 (Washington State Probe-1, Cayman Chemical; Liu et al., 2011) at a concentration of 100 pM in standard buffer pH 7.4 (composition: 20 mM HEPES, 120 mM NaCl, 2 mM KC1, 2 mM CaCl ₂ -2H ₂ O, 1 mM MgCl ₂ -6H ₂ O, 5 mM glucose). After a 30-minute incubation at 37°C, the supernatant was removed and the cells were treated with the vehicle (1% DMSO), diallyl disulfide (DADS) at a concentration of 100 pM as a reference compound since it is a known H ₂ S donor, or with the compound of the invention (compound of formula II) at concentrations of 10 pM, 100 pM and ImM for the MCF-7 and MCF-10A cell lines, and at concentrations of 10 pM, 500 pM and ImM for the AsPc-1 and MIAPaCa-2 cell lines. The fluorescence, and thus the amount of H ₂ S released within the cells, was monitored for 45 minutes at the wavelengths of kex 465 and kern 515 nm by means of a spectrofluorimeter.

As can be seen from Figures 10, 11 and 12, the compound according to the invention showed a significant release of H ₂ S within the AsPc-1, MIAPaCa-2, and MCF-7 cancer cells, whereas no increase in intracellular H ₂ S was detected in MCF-10A cells (non-tumor cells).

Example 9: Assessment of the activity of the compounds of the invention on the cell cycle

In order to analyse the effects of the compound of the invention on the cell cycle, the inventors determined the apoptotic markers caspase 3/7 and annexin V. Briefly, MCF-7 cells (breast adenocarcinoma) were plated and after 24 hours the plating medium was replaced with “fresh” medium and the cells were treated for 72 hours with the vehicle (1% DMSO) or with the compound of the invention (compound of formula II) at concentrations of 10 pM, 20 pM and 100 pM. Cell cycle distribution was analysed by a flow cytometer using the Muse™ Cell Cycle Kit (EMD Millipore Bioscience) and following the manufacturer's protocol. MCF-7 cells (breast adenocarcinoma) were plated in 6-well cell culture plates at a density of 5xl0 ⁵ cells per well, and after incubation with the treatments (72 hours) the cells were washed with phosphate buffer (PBS), mechanically detached, and fixed in 70% ethanol at -20°C for at least 3 hours. The suspension was then washed with PBS and IxlO ⁶ cells were incubated with 200 pl of the “cell cycle reaction” solution supplied by the kit for 30 minutes at 37°C in the dark. The cell suspension was analysed using the Muse™ Cell Analyzer (Millipore) tool.

The activity of the caspase 3/7 enzymes was measured by a flow cytometer using the “Muse™ Caspase 3/7 Activity Kit” (EMD Millipore Bioscience), according to the manufacturer’s protocol. MCF-7 cells (breast adenocarcinoma) were plated in 24-well cell culture plates at a density of IxlO ⁵ cells per well, and after the treatments (72 hours) the cells were mechanically detached, washed with PBS, and resuspended at a concentration of 5x10 ⁵ cells/ml in the buffer supplied by the kit. Then, 5 pl of the solution called “Muse™ Caspase- 3/7 Reagent working solution” were added to 50 pl of the cell suspension and incubated at 37°C for 30 minutes. Finally, a further 150 pl of the solution called “Muse™ Caspase 7- AAD working solution” was added and incubated at 37°C for 5 minutes. The cells thus labelled were read by the Muse™ Cell Analyzer.

Annexin V expression was measured by a flow cytometer using the “Muse™ Annexin V Kit” (EMD Millipore Bioscience), according to the manufacturer’s protocol. MCF-7 cells (breast adenocarcinoma) were plated in 24-well cell culture plates at a density of IxlO ⁵ cells per well, and after the treatments (72 hours) the cells were mechanically detached, washed with PBS, and resuspended at a concentration of 5xl0 ⁵ cells/ml in the buffer supplied by the kit. Then, 100 pl of the solution called “Muse™ Annexin V” were added to 100 pl of the cell suspension and incubated at 37°C for 30 minutes. The cells thus labelled were read by the Muse™ Cell Analyzer.

The compound of the invention showed the ability to cause H2S intracellular release in a concentration-dependent manner in all cell models tested. The ability to inhibit cell viability after 72 hours of incubation was assessed in the different tumor cell lines, where the compound of the invention caused approximately 100% cell viability inhibition at a concentration of ImM in AsPc-1 cells, 5mM in MIAPaCa-2 cells, 500 pM in MCF-7 cells, and 1 mM in MCF-10A cells, showing that the additional ability to donate H2S resulted in a synergistic enhancement of the anti-proliferative activity compared to the native metformin molecule which, in all cell lines tested, resulted in approximately 50% cell viability reduction at much higher concentrations (5mM or higher) (Figures 6-9). The breast tumor model (MCF-7 cell line) related to the non-tumor reference (MCF-10A cell line) made it possible to highlight that the anti-proliferative activity potency of the compound of the invention (compound of formula II) in healthy cells (approximately 170 pM) is significantly higher than that found in cancer cells (approximately 20 pM), pointing out a certain selectivity for diseased tissue compared to healthy tissue (Figure 14).

Furthermore, the compound of the invention showed a significant enhancement in the antiproliferative potency in MCF-7 breast adenocarcinoma, even when compared with native metformin which exhibited lower potency parameters (approximately 5 mM).

The ability of the compound of the invention to alter the cell cycle compared to the control was also tested in MCF-7 breast cancer cells (Figure 15). The results of these experiments have shown that the compound of the invention results in a significant reduction in the G0/G1 phase and an increase in the number of cells in the G2/M phase. Lastly, the compound according to the invention is capable of increasing cellular apoptosis, measured as an increase in annexin V and caspase 3/7 (Figures 16 and 17).