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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/28—Phosphorus compounds with one or more P—C bonds
  • C07F9/54—Quaternary phosphonium compounds
  • C07F9/5442—Aromatic phosphonium compounds (P-C aromatic linkage)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• Triphenylphosphonium biguanide analogues their method of preparation and use as drugs Field of Art
• the invention relates to novel biguanide derivatives with high effectivity against type 2 diabetes mellitus and pancreatic cancer.
• Type 2 diabetes mellitus is a metabolic disease affecting a growing number of subjects in industrialised countries. During last decades the number of patients suffering from T2DM increased in an unprecedented manner. It is expected that around 2030 the number of patients suffering from this disease will double. At present, the disease can be considered a civilization epidemic. T2DM is resistant to insulin treatment. The most commonly used drug against this pathology worldwide is metformin, prescribed to tens of millions of patients. Metformin decreases the level of glucose, which is released by glycogenolysis or synthesized by gluconeo genesis in hepatic cells. Metformin is a biguanide drug with low risk of side effects and complications. Oxidative phosphorylation- related processes are affected also by other clinically relevant biguanides such as fenformin, buformin, proguanil and cycloguanil or chlorhexidine.
• biguanides such as fenformin, buformin, proguanil and cycloguanil or chlorhexidine.
• T2DM is related to neoplastic diseases (Richardson LC, Pollack LA. Nat Clin Pract Oncol. 2005, 2, 48-53), in particular to pancreatic cancer (Bosetti C et al. Ann Oncol 2014 25, 2065-2072. Rahman A. Lancet Oncol 2014 15, e420).
• T2DM is sometimes considered to a "precarcinogenic" stage of pancreatic cancer (Eijgenraam P et al. Br J Cancer 2013, 109, 2924-2932).
• Pancreatic carcinoma is an extremely hard to treat type of cancer, with basically resection being an option. This is possible only in a limited group of patients, depending on the location and stage of the carcinoma.
• Pancreatic cancer belongs to neoplastic diseases with the highest number of deaths.
• One of the complicating factors in pancreatic cancer stems from the fact that up to 90 % of patients is are positive for the oncogene Ras, that causes malignant transformations and considerably complicates therapy.
• Metformin the most commonly prescribed drug against T2DM, has diabetes mellitus type 2 patients was found to suppress pancreatic cancer, although the effect is not very high and the compound is efficient at high, milimolar concentrations (Gong J et al. Front Physiol 2014, 5, 426).
• T2DM as well as pancreatic cancer are very serious conditions that are at present almost unbeatable and the incidence of which is constantly increasing. There is a strong need to find novel agents and novel medical approaches against these diseases.
• the present invention provides a new generation of substances derived from the basic structure of alkylated biguanides (buformin, metformin and fenformin) that show 3-4 orders of magnitude higher effect against pancreatic cancer and T2DM compared to metformin, while not causing toxic effects in experimental animals.
• the present invention thus provides triphenylphosphonium biguanide analogues of the general formula I, whereas the general formula I shall be considered to include also resonance (isomeric) structures and pharmaceutically acceptable salts, protonated biguanide forms as well as free bases
• Z is a linear hydrocarbyl chain selected from alkylene, alkenylene or alkynylene, containing 2 to 20 carbon atoms, preferably 4 to 14 carbon atoms, more preferably 8 to 12 carbon atoms, whereas optionally one or more carbon atoms couples in the hydrocarbyl chain may be replaced by one or more 5-membered or 6-membered aromatic rings or heteroaromatic rings containing the heteroatoms O, S and/or N, preferably phenylenes or pyridylenes, and/or one or more carbon atoms in the hydrocarbyl chain may be replaced by one or more heteroatoms selected from O, S, NH, and whereas the hydrocarbyl chain can be unsubstituted or substituted by one or more substituents selected independently from the group comprising C1-C4 alkyl, N(H or C1-C4 alkyl) 2 , wherein alkyls are the same or different, phenyl, benzyl, OH, SH,
• Rl, R2, R3, R4, R5, R6, R7 is a substituent of general formula II
• X " is a pharmaceutically acceptable anion, in particular anion of inorganic or organic acid, particularly suitable are CI " , Br " , ⁇ , sulphate, mesyl, acetate, formiate, succinate,
• Y " is a pharmaceutically acceptable anion, in particular anion of inorganic or organic acid, particularly suitable are CI " , Br " , ⁇ , sulphate, mesyl, acetate, formiate, succinate.
• Rl is a substituent of general formula II
• Rl and R2 are substituents of general formula II
• R3 is a substituent of general formula II
• C6-C10 aryl is 4-chlorfenyl.
• (Cl-C6)alkyl(C6-C10)aryl is phenylethyl or 2-(2-acetoxy-4,6-dimethylphenyl)- propyl.
• the present invention further provides a method for preparation of the compounds of general formula I, wherein in the first step, a compound of general formula III
• T is halogen, mesyl, tosyl or other cleavable group and Z has the meaning as defined above,
• triphenylphosphine preferably in dimethylformamide (DMF)
• DMF dimethylformamide
• triphenylphosphonium hydrocarbyl biguanide derivative of general formula I is subsequently condensed with 2-cyanoguanidine, preferably in DMF, yielding triphenylphosphonium hydrocarbyl biguanide derivative of general formula I.
• the compounds of the present invention do not show toxic effects on non-malignant cells, hence, they are selective in killing the cells of pancreatic carcinoma.
• the compounds of the present invention highly effectively inhibit growth of experimental pancreatic carcinoma.
• Antidiabetics activity is characteristic in that these compounds decrease glucose levels in circulating blood.
• glucose levels are increased mainly as a result of glucose release from its storage source, which is glycogen in hepatocytes, by glycogenolysis, and also as a result of gluconeogesis (formation of glucose).
• Metformin acts on these processes and thus decreases glucose levels in patients suffering from diabetes mellitus type 2.
• the compounds of the present invention decrease glucose levels in concentrations which are 3 orders of magnitude lower than the effective concentrations of metformin.
• Object of the present invention are thus compounds of general formula I for use as medicaments, in particular for use in a method of treatment of diabetes mellitus type 2 and/or pancreatic carcinoma.
• Object of the present invention is use of compounds of general formula I for preparation of a medicament for the treatment of diabetes mellitus type 2 and/or pancreatic carcinoma.
• object of the present invention is a method of treatment of mammals, including human, in which one or more compounds of general formula I is administered to a subject suffering from diabetes mellitus type 2 and/or pancreatic carcinoma.
• Object of the present invention is also a pharmaceutical preparation containing at least one compound of general formula I and at least one pharmaceutical auxiliary substance, such as a carrier, a solvent, a filler, a colorant, a binder, etc.
• pharmaceutical auxiliary substance such as a carrier, a solvent, a filler, a colorant, a binder, etc.
• Fig. l Concentration dependence of growth inhibition of PANC-1 cells as a response to studied compounds and metformin using the crystal violet method.
• Fig. 2 IC 50 values for compound 7 and metformin for cancer cells PANC-1 and non- malignant BJ fibroblasts.
• FIG. 3 A - The comparison of the effect of different concentrations of compound 7 on the growth curve of PANC-1 cells acquired by the xCellingence method.
• B Concentration and time dependence of growth curve slope.
• Fig. 4. IC 50 values for compounds 7, 15 and metformin determined from growth curves.
• Fig. 5. Annexin test of apoptosis for pancreatic cancer cell lines using compound 7 and metformin.
• Fig. 6 The comparison of relative level of active caspase-3, a marker of apoptosis detection for control cells, cells treated with compound 7, and cells treated with metformin. Treatment with staurosporin is used as positive control.
• Fig. 7 Inhibition of experimental pancreatic cancer with compound 7 compared with the control.
• Fig. 8 Concentration dependence of inhibition of cellular respiration via mitochondrial complex I by compounds 7, 9, 10 and metformin.
• Fig. 9 The effect of compound 7 and metformin on mitochondrial membrane potential in a pancreatic cell line PANC- 1.
• Fig. 10 The effect of compound 7 and metformin on formation of reactive oxygen species.
• Fig. 11 Suppression of glucose level in HepG2 cells by compound 7 and metformin.
• 1,10-Dibromodecane (5.72 g, 19.063 mmol) and triphenylphosphine (1 g, 3.813 mmol) were dissolved in DMF (5 ml) and the mixture was heated at 90 °C for 12 h. The reaction mixture was cooled to room temperature, dissolved in minimal amount of dichloromethane (5 ml) and added drop-wise to cold (0 °C) diethylether (200 ml).
• IR (KBr pellet): v 3053, 3040, 3005, 2990, 2938, 2860, 2796, 2212, 2006, 1931, 1821, 1685, 1610, 1587, 1575, 1483, 1459, 1435, 1333, 1252, 1186, 1110, 995, 786, 751, 722, 692.
• IR (KBr pellet): v 3409, 3051, 3007, 2935, 2864, 2739, 2417, 2215, 2002, 1924, 1832, 1783, 1614, 1586, 1484, 1462, 1438, 1318, 1189, 1162, 1113, 996, 751, 723, 691.
• Biguanide derivative of the formula 8 was prepared using the same procedure as described in example 7.
• IR (KBr pellet): v 3315, 3180, 3052, 2935, 3861, 2804, 1690, 1631, 1587, 1559, 1503, 1438, 1113, 996, 750, 723, 691.
• Biguanide derivative of the formula 10 was prepared using the same procedure as described in example 9.
• IR (KBr pellet): v 3311, 3160, 2926, 2853, 1636, 1533, 1507, 1438, 1398, 1337, 1162, 1113, 996, 784, 723, 691.
• Triethylamine (5mL) and (Boc) 2 0 (2.160 g, 9.181 mmol) were added to the solution of /?-aminoethylbenzoic acid hydrochloride (l.OOOg, 4.959 mmol) in dichloromethane (15 ml). Resulting mixture was stirred for 48 hours at laboratory temperature. Reaction mixture was diluted with diethylether (300 ml) and washed with citric acid (15%, 3 x 50 ml). Organic layer was dried over MgS0 4 and filtered.
• pancreatic cancer cell lines Using five different pancreatic cancer cell lines, we show that the IC 50 values are for the new compounds up to 4 orders of magnitude lower that for the known compound metformin, which is currently undergoing clinical testing as an efficient drug against pancreatic cancer. The highest anti-cancer effects were found for compound 7, which was therefore studied in further experiments.
• Effective and safe anti-cancer agents should be selective for malignant cells and, at the same time, non-toxic to normal cells.
• IC50 values for compound 7 determined by the crystal violet assay is obvious that compound 7 suppresses growth of PANC-1 cells at considerably lower concentration compared to non-tumor line (BJ fibroblasts) (Fig. 2), and should therefore suppress only growth of tumours without deleterious effect on healthy cells, using appropriate doses.
• Figure 3 A depicts growth curves of PANC-1 cells under control conditions and in the presence of compound 7 in the cultivation medium (the arrow shows time of supplementation). Lower slope of the curve indicates that compound 7 suppresses growth and that, at higher concentrations, it even causes cell death, which is suggested by negative slope of the growth curve ( Figure 3B).
• Example 20 This example shows whether the new compounds according to the invention trigged programmed cell death, i.e. apoptosis.
• the pancreatic cancer cell lines PANC-1 and MiaPaCa-2 were used.
• Apoptotic cell death was determined using annexin V, which is a protein with affinity for phosphotidylserine that is externalized during apoptosis. Under these conditions, annexin V binds to the phospholipid (Weber T et al (2003) Mitochondria play a central role in apoptosis induced by oc-tocopheryl succinate, an agent with anticancer activity. Comparison with receptor-mediated pro-apoptotic signaling. Biochemistry 42, 4277-4291).
• annexin V used for this assay was fluorescently labeled, the extent of apoptotic cell death can be quantified using flow cytometry.
• Fig. 5 shows that, compared to compound 7 that efficiently kills the cancer cells, metformin is inefficient at the same concentration levels.
• Results documented in Example 20 were verified by evaluation of caspase-3 activation in PANC-1 cancer cells exposed to compound 7 or metformin.
• Caspase-3 is a key protein of the apoptotic cascade, whose activation causes cell death. Considerable activation of capase-3 was observed after 48 h of incubation of the cells in the presence of compound 7. Metformin used at 1,000-fold higher concentration was inefficient (Fig. 6). Staurosporin was used here as a positive control, since it is an efficient apoptosis inducer.
• Compound 7 was applied twice per week at doses some 20- and 40-fold lower that those published for metformin (84 nmol/g for MiaPaCa-2 cell-derived tumours and 126 nmol/g for PANC-1 cell-derived tumours), at which level the known compound suppresses pancreatic tumours (Kisfalvi K et al. Metformin inhibits the growth of human pancreatic cancer xenografts. Pancreas 2013, 42, 781-785).
• Compound 7 was applied orally using the gavage technique, which allows administration of the agent into the digestive system of an animal, which allows its uptake into the circulation that then takes the agent to target tissues (cells).
• Fig. 7 documents high anti-cancer activity of compound 7 that suppresses tumour growth in spite of being applied at lower doses compared to the metformin-treated animals (compared to animals with PANC-1 cell-derived tumours treated with metformin, compound 7 suppressed tumour growth by the factor of 54 (12.17 x 40); for MiaPaCa-2 cell-derived tumours this factor was 93 (35/15 x 40).
• An important aspect of these experiments was that compound 7 was not toxic, as documented on the bases of animal behaviour and no weight loss.
• metformin also suppresses respiration via mitochondrial complex I, which is one of the mechanisms of the anti-cancer activity of the agent ((Wheaton, W. W., et al. (2014). Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis. Elife 3: e02242.).
• Fig. 8 shows that compound 7 suppresses MIaPaca-2 cell complex I respiration with IC 50 of 3 ⁇ and that of PaTu 8902 with IC 50 of 7.5 ⁇ .
• Metformin started to inhibit complex I respiration at levels of about 1 mM, i.e. it was again some 3 orders of magnitude less efficient than compound 7.
• Example 24 Biguanide analogues induce their activity towards cancer cells by targeting mitochondria. We therefore studied their effect on mitochondrial potential that is a prerequisite for proper mitochondrial function, including efficient import of mitochondrial proteins from the cytoplasm. We followed the effect on mitochondrial potential using the fluorescent probe tetramethylrhodamine methyl ester (TMRM) that loses red fluorescence upon a decrease (dissipation) of mitochondrial potential (Rohlena J et al. Mitochondrial ⁇ targeted oc- tocopheryl succinate is antiangiogenic: Potential benefit against tumor angiogenesis but caution against wound healing. Antiox Redox Signal 2011, 15, 2923-2935).
• TMRM fluorescent probe tetramethylrhodamine methyl ester
• the studied PANC-1 cells revealed important loss of mitochondrial potential after 24 h incubation with compound 7, whereas when 5 ⁇ concentration was used, a complete loss of detectable potential occured. Metformin used at 200-fold higher concentration showed no effect on mitochondrial potential (Fig. 9).
• Metformin is the world most frequently subscribed agent against type 2 diabetes mellitus. Since the new compounds according the invention are considerably more efficient against pancreatic cancer compared to metformin, we also tested them for a potential effect against aspects of type 2 diabetes mellitus.
• One aspect of diabetes is increased level of glucose in circulation due to increased formation of glucose in hepatocytes by the process of guconeogenesis as well as by release of glucose from glycogen due to glycogenolysis. Therefore we tested the level of glucose using cultured hepatic cell line HepG2 in the presence of metformin and compound 7 using a published protocol (Magni F et al. Determination of serum glucose by isotope dilution mass spectrometry: candidate definitive method. Clin. Chem.
• the compounds of the present invention represent a new generation of medicaments for treatment of diabetes mellitus type 2 and pancreatic carcinoma, with high activity and without toxic side effects.

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