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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
  • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
  • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
  • C07F15/0086—Platinum compounds
  • C07F15/0093—Platinum compounds without a metal-carbon linkage
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/24—Heavy metals; Compounds thereof
  • A61K33/243—Platinum; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
• • 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
  • C07F13/00—Compounds containing elements of Groups 7 or 17 of the Periodic Table
• • 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
  • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
  • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
  • C07F15/0086—Platinum compounds

Definitions

• the invention relates to platinum-based antitumor drugs and more particularly relates to diiodidoplatinum(II) complexes of the general formula cis-[PtI 2 L 2 ], wherein L represents an N-donor heterocyclic ligand based on 1H-imidazole, and their use for the preparation of medicaments for the treatment of multiple myeloma.
• Cisplatin cis-diammine-dichloridoplatinum(II) complex; formula A
• Cisplatin has been a platinum-based anticancer chemotherapeutic agent in use for over forty years (Rosenberg et al., Nature 205 (1965) 698; Kelland, Nat. Rev. Cancer 7 (2007) 573).
• its clinical use is associated with numerous side effects (e.g. nephrotoxicity or myelosuppression)
• the problems include also the resistance of several types of tumors towards cisplatin, both natural and acquired resistance.
• the possibility of improving the activity and overall pharmacological profile of cisplatin and its clinically used derivatives e.g.
• carboplatin diammine-cyclobutane-1,1 ⁇ -dicarboxylatoplatinum(II) complex
• oxaliplatin (1R,2R- diaminocyclohexane-oxalatoplatinum(II) complex)
• platinum-based compounds e.g. highly efficient multi-target Pt(IV) complexes; Gibson, J. Inorg. Biochem. 217 (2021) 111353
• compounds derived from other transition metals derived from other transition metals
• compounds derived from other transition metals derived from other transition metals
• hybrid formulations for targeted delivery of platinum-based drugs Wang a Guo, Chem. Soc. Rev.42 (2013) 202).
• Multiple myeloma which is an incurable disease, is also referred to as plasma cell myeloma. It is a cancer of plasma cells located in the bone marrow, causing osteolytic lesions or osteoporosis, and in most cases it is associated with the presence of M-protein/paraprotein in the blood serum. Treatment is initiated if the patient meets the criteria for multiple myeloma. Currently, there are two diagnostic criteria for multiple myeloma.
• the first criteria are approved by the World Health Organization (WHO).
• the second criteria were issued by the International Myeloma Working Group (IMWG; Br. J. Haematol.121 (2003) 749).
• IMWG International Myeloma Working Group
• IMWG International Myeloma Working Group
• There have been advances in the treatment of symptomatic multiple myeloma particularly with the advent of proteasome inhibitors - bortezomib, carfilzomib and ixazomib (Wong et al., Cancers 12 (2020) 2203) and immunomodulatory agents (such as lenalidomide). Treatment starts with chemotherapy and the main treatment agents are bortezomib and dexamethasone.
• Bone marrow stromal cells proliferate and survive in the bone marrow through physiological and functional interactions with bone marrow stromal cells and the surrounding bone marrow microenvironment (Bianchi and Munshi, Blood 125 (2015) 3049; Abe, Int. J. Hematol.94 (2011) 334; Meads et al., Clin. Cancer Res.14 (2008) 2519).
• Bone marrow stromal cells produce inflammatory cytokines such as IL-6 and, in myeloma cells, regulate the expression of cell cycle inhibitors - cyclin-dependent kinases p21WAF1 and p27Kip1, anti-apoptotic members of the Bcl-2 family or transport ABC proteins (Furukawa and Kikuchi, Int.
• Interleukin-6 is a pleiotropic cytokine, a growth factor that is essential for skeletal homeostasis (Harmer et al., Front. Endocrinol.9 (2019) 788), and functions as an anti-apoptotic factor in multiple myeloma (Klein et al., Blood 85 (1995) 863; Lichtenstein et al., Cell Immunol.162 (1995) 248).
• IL-6 Since the identification of IL-6 has been used in the past as a major growth factor for myeloma cells, there is a group of human myeloma cell lines that have been derived from extramedullary cultures of primary myeloma cells expressing IL-6 from patients with multiple myeloma (Zhang et al., Blood 83 (1994) 3654).
• the myeloma cell line U266 (U266B1) is independent of IL-6, which blocks Interleukin- 6-inhibited cell growth, but U266 cells express IL-6 (Ingersoll et al., Med. Chem.7 (2011) 473).
• the present invention provides electroneutral cis-diiodido complexes of platinum(II), said complexes having the general formula C, wherein: - R1 is independently on each occurence selected from the group consisting of hydrogen and C1-C4 alkyl; - R2 is independently on each occurence selected from the group consisting of C1–C4 alkyl, phenyl and phenyl-substituted C1–C2 alkyl, wherein the phenyl ring may be unsubstituted or substituted b y halogen or C1-C4 alkoxy group; - provided that if R2 is methyl, R1 is not hydrogen; optionally in the form of pharmaceutically acceptable solvates.
• R2 is fluorophenyl.
• the present invention provides electroneutral cis-diiodido complexes of platinum(II), said complexes having the general formula C, wherein: - R1 is independently on each occurence selected from the group consisting of hydrogen and C1-C4 alkyl; - R2 is independently on each occurence selected from the group consisting of C1–C4 alkyl, phenyl and phenyl-substituted C1–C2 alkyl, wherein the phenyl ring may be unsubstituted or substituted by halogen or C1-C4 alkoxy group; optionally in the form of pharmaceutically acceptable solvates; for use as medicaments, in particular for use in the treatment of tumor diseases, and most preferably for use in the treatment of multiple myeloma.
• the invention also relates to the preparation of the complexes of the general formula C, by a procedure starting from tetrachloridoplatinate(II), wherein tetrachloridoplatinate(II) is reacted with an alkali metal iodide to produce tetraiodidoplatinate(II) in situ, and the tetraiodidoplatinate(II) is then subjected to a reaction with an equimolar amount of the corresponding 1H-imidazole derivative (ligand precursor).
• the reaction for the preparation of complexes of general formula C is preferably carried out at laboratory temperature for 12 to 36 h, preferably for 24 h.
• the reaction is preferably carried out in a solvent selected from the group consisting of primary alcohol, water, and their mixtures.
• the product is precipitated or crystallized and separated from the mother liquor by filtration or centrifugation.
• the separated product can preferably be washed with water and a solvent selected from the group consisting of primary alcohol, secondary alcohol, acetone, n-hexane, diethyl ether, and their mixtures.
• the isolated product can then be dried in a desiccator or under an infrared lamp.
• the compounds of formula C may be in the form of crystal solvates.
• Crystal solvates can be described, for example, by the formula cis-[PtI 2 L 2 ] ⁇ nSolv, wherein L is a substituted 1H- imidazole, the symbol n indicates the number of crystal-bound particles, typically 1 to 4, and Solv is a solvent molecule.
• the solvent molecule is preferably selected from the group consisting of water, primary alcohol, secondary alcohol, acetone, n-hexane, diethyl ether, and mixtures thereof.
• the invention relates to a pharmaceutical preparation containing (a therapeutically effective amount) of at least one diiodidoplatinum(II) complex of general formula C, and at least one excipient. Excipients typically include fillers, binders, solvents, glidants, disintegrants, stabilizers, and/or taste and smell corrigents.
• the pharmaceutical preparation is preferably a liquid preparation, for example in the form of an injection or infusion solution. Brief Description of Drawings Fig.
• Fig.2 shows the number of viable cells of the myeloma line U266 (U266B1) after application of 1 ⁇ M, 2 ⁇ M and 3 ⁇ M concentrations of complexes 1 to 7.
• the 1 ⁇ M concentration caused a decrease in the proportion of viable cells below 20 % after exposure to complex 7, while a similar reduction in the proportion of viable cells occurred at a concentration of 2 ⁇ M of complexes 5, 6 and 7.
• a proportion of living cells lower than 10 % was detected after using a concentration of 3 ⁇ M of complexes 3 to 7.
• Fig.3 shows the number of viable cell of the myeloma line KMS12-PE after application of 1 ⁇ M, 2 ⁇ M and 3 ⁇ M concentrations of complexes 1 to 7. About and below 10 % viable cells were detected after treatment by 1 ⁇ M concentration of complexes 6 and 7, while a similar reduction in viable cells occurred after application of 2 ⁇ M concentration of complexes 2 to 7. A concentration of 3 ⁇ M of all tested complexes 1 to 7 resulted in less than 10 % viable cells. Fig.
• FIG. 4 shows the number of viable cells of the myeloma lines U266 (U266B1) and KMS12-PE, and the fibroblast line HS-5 after application of 1 ⁇ M, 2 ⁇ M and 3 ⁇ M concentrations of complex 6.2 ⁇ M and 3 ⁇ M concentrations of complex 6 caused a decrease in the proportion of viable cells in both tested myeloma lines below 20 % and 10 %.
• the proportion of viable cells of the HS-5 line is around 80 % after the application of 1 ⁇ M concentration, and it is reduced to 50–60 % after the application of 2 ⁇ M and 3 ⁇ M concentrations of complex 6.
• Fig. 5 is the ratio of cell cycle phases in the myeloma cell line U266 (U266B1).
• Fig.7 shows the number of viable cells of the myeloma line U266 (U266B1), which after the application of 2 ⁇ M concentration of complex 6 is reduced to 43.3 % by determination of caspases 3 and 7 (37.4 % Annexin V/7-AAD) and after the application of 3 ⁇ M concentration of complex 6 to 7.0 % by determination of caspases 3 and 7 (9.7 % Annexin V/7-AAD) when compared to untreated cells (DMF - 82.4 % by determination of caspases 3 and 7 (80.0 % Annexin V/7 -AAD).
• the numbers of viable cells are significantly lower after exposure to 3 ⁇ M (p ⁇ 0.001 for Annexin; p ⁇ 0.05 for caspases 3 and 7) concentrations of complex 6, compared to the numbers of viable cells cultured in medium with control DMF. Exposure to 1 ⁇ M concentrations has no effect on apoptosis.
• Fig. 8 shows the number of viable cells of the myeloma line KMS12-PE, which after the application of 1 ⁇ M concentration of complex 6 is reduced to 38.2 % by determination of caspases 3 and 7, compared to 86.4 % of untreated viable cells (DMF), and leads to apoptosis of the treated myeloma cells.
• Fig. 9 shows the cell cycle phase ratio of the myeloma cell line U266 (U266B1) and the fibroblast cell line HS-5 cultivated in the co-culture system. Exposure of cells to 2 ⁇ M and 3 ⁇ M concentrations of complex 6 leads to the induction of apoptosis by increasing the number of cells in the sub-G1 phase of the cell cycle.
• a 1 ⁇ M concentration of complex 6 is not effective enough to induce apoptosis, but it is sufficient to arrest the cell cycle in the G1 phase.
• Fig.10 shows the ratio of the cell cycle phases of cells of the myeloma line KMS12-PE and cells of the fibroblast line HS-5 co-cultured in a co-culture system.
• a 1 ⁇ M concentration of complex 6 causes an increase in the number of cells in the sub-G1 phase of the cell cycle, 2 ⁇ M and 3 ⁇ M concentrations appear to be toxic to co-cultured cells.
• Fig. 11 shows the number of viable cells in the co-culture of the myeloma line U266 (U266B1) and the fibroblast line HS-5, which is reduced after the application of 2 ⁇ M and 3 ⁇ M concentrations of complex 6 when compared to untreated cells.
• Fig.12 shows the number of viable cells cultured in the co-culture of the myeloma line KMS12-PE and the fibroblast line HS-5, which is reduced after the application of 1 ⁇ M concentration of complex 6, when compared to untreated viable cells (DMF) (p ⁇ 0.01 for Annexin).
• a 1 ⁇ M concentration of complex 6 causes apoptosis in the treated co-cultured cell lines.
• 2 ⁇ M and 3 ⁇ M concentrations of complex 6 lead to a type of cell death other than apoptosis or caspase-independent cell death (specifically caspases 3 and 7).
• Example 1 Preparation and characterization of complexes 1 to 7
• Commercially available organic compounds 1-methyl-1H-imidazole (L 1 ), 1-benzyl-1H-imidazole (L 2 ), 1-benzyl-2-methyl-1H-imidazole (L 3 ), 1-phenyl-1H-imidazole (L 4 ), 1-(4-methoxyphenyl)-1H-imidazole (L 5 ), 1-(4-fluorophenyl)-1H-imidazole (L 6 ) and 1-(4-chlorophenyl)-1H-imidazole (L 7 ), which were purchased from Sigma-Aldrich (Prague, Czech Republic), were used for the syntheses of the complexes.
• Complex 3 Yellow solid. Yield 85 %. Elemental analysis: calculated for C 22 H 24 I 2 N 4 Pt: C, 33.31; H, 3.05; N, 7.06 %; found: C, 33.64; H, 3.35; N, 7.48 %.
• IR ATR, ⁇ , cm -1 ): 468w, 543w, 614w, 643w, 738w, 803w, 837m, 966w, 1011w, 1063w, 1127w, 1149w, 1221m, 1304w, 1519s, 3110m.
• Example 2 In vitro cytotoxicity of the complexes 1 to 7 towards multiple myeloma
• U266 U266B1
• KMS12-PE the human myeloma cell lines U266 (U266B1) and KMS12-PE, and the non-tumor fibroblast cell line HS-5
• MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
• the test detects viable, metabolically active cells that, unlike dead cells, enzymatically reduce the MTT dye to its purple formazan. The number of metabolically active cells then corresponds to the amount of reduced MTT, i.e.
• the myeloma cell line U266 (U266B1) is a suspension line. It was purchased from the American Type Cell Collection (ATCC ® TIB-196TM). It was created from the peripheral blood of a 53-year-old man with multiple myeloma secreting immunoglobulin IgE (source dsmz.de). Myeloma cells of the line U266 (U266B1) express Interleukin-6 (Ingersoll et al., Med. Chem. 7 (2011) 473). Morphologically, the cells are single or grouped round to polygonal cells in suspension.
• the myeloma cell line KMS12-PE is a suspension line. It was obtained from the JCRB - Japanese Cancer Research Resources Bank (National Institute of Biomedical Innovation, Osaka, Japan) under the designation JCRB0430. It was created from the pleural effusion of a 64-year-old woman with refractory end-stage multiple myeloma (non-immunoglobulin-producing) after combination chemotherapy. Morphologically, the cells are small round cells that grow in cell suspension individually or in small clusters (source dsmz.de).
• the HS-5 cell line (ATCC ® CRL-11882TM) purchased from American Type Cell Collection was derived from transformed fibroblasts obtained from the bone marrow of a healthy 30-year-old male. These are adherent cells (source dsmz.de). Cells of the myeloma line U266 (U266B1) were cultured in RPMI-1640 medium supplemented with 15% fetal bovine serum, 1% antibiotics Penicillin-Streptomycin, 1% L-glutamine and 100 mM sodium pyruvate, according to the supplier's instructions (ATCC) - in incubator at 37 °C, 100% humidity and 5% CO 2 atmosphere.
• ATCC The HS-5 cell line (ATCC ® CRL-11882TM) purchased from American Type Cell Collection was derived from transformed fibroblasts obtained from the bone marrow of a healthy 30-year-old male. These are adherent cells (source dsmz.de). Cells of the myeloma line U266 (
• Cells of the myeloma line KMS12-PE and the fibroblast line HS-5 were cultured under the same conditions as the myeloma line U266, but with only 10% fetal bovine serum.
• Tested complexes 1 to 7 were dissolved in N,N'-dimethylformamide (DMF) to prepare a 50.0 mM stock solution.
• the stock solution was diluted with medium to concentrations of 1 ⁇ M, 2 ⁇ M and 3 ⁇ M.
• U266 (U266B1) and KMS12-PE myeloma cell suspensions were pipetted into a ninety-six-well microtitre plate, and at 35–40% cell confluence, 1 ⁇ M, 2 ⁇ M, and 3 ⁇ M concentrations of complexes 1 to 7 were added to the cells.
• the treated myeloma cell suspensions were subsequently incubated for 24 h (37 °C, 100% humidity, 5% CO 2 ). At the same time, the effect of complexes 1 to 7 on the cell suspension of fibroblasts was tested.
• RPMI medium with 10% FBS was used as a negative control, and 0.015% DMF in the medium (amount of solvent in which the tested complexes were dissolved) was used as another control.
• MTT solution was added to the mixtures, followed by incubation for 4 h at 37 °C.
• the reaction was stopped by the application of 10% SDS, and after another 24 h of incubation of the treated cell suspensions at 37 °C, spectrophotometric analysis was performed with a Labysystems Multiskan RC ELISA device at a wavelength of 570 nm. Finally, cell viability was calculated for DMF and for the three concentrations of each of the tested complexes 1 to 7.
• Three independent experiments were performed from newly passaged cells.
• Table 1 Viability observed in tested cell lines U266B1, KMS12-PE and HS-5 for the complexes 1–7
• Example 3 Detection of apoptosis and cell cycle in myeloma cell lines The effects of complex 6 on the course of the cell cycle of myeloma cells differing in the expression of interleukin-6 (IL-6) were investigated after 24 h of exposure. Cell cycle analysis was performed by flow cytometry of cells stained with propidium iodide (PI).
• PI propidium iodide
• Myeloma cells of the U266 (U266B1) line expressing IL-6 and the KMS12-PE line without IL-6 expression were treated with 1 ⁇ M, 2 ⁇ M and 3 ⁇ M concentrations of complex 6 for 24 h, fixed with 96% ice-cold ethanol, washed with ice-cold PBS with 2% fetal bovine serum and subsequently incubated for 30 min at room temperature with a PBS solution containing RNAse A (0.2 mg/ml). After addition of PI, cell cycle analysis was performed by flow cytometry (BD FACSVerse, BD Biosciences, San Jose, CA, USA).
• Apoptosis detection was performed by staining for Annexin V/7-AAD and determining the activities of caspases 3 and 7. While Annexin V provides a very sensitive method for detecting cell apoptosis, 7-aminoactinomycin D (7-AAD) is used to detect necrotic or late apoptotic cells.
• Annexin V/7-AAD staining was used to semi-quantify viable, apoptotic and necrotic cells after 24 h treatment with 1 ⁇ M, 2 ⁇ M and 3 ⁇ M concentrations of complex 6 on cell suspensions of myeloma line U266 (U266B1) and line KMS12-PE.
• the treated cells were harvested after 24 h, centrifuged to form a cell pellet, which was stored on ice.
• Binding buffer 140 mM NaCl, 4 mM KCl, 0.75 mM MgCl 2 , and 10 mM HEPES
• Annexin V, 7-AAD, and CaCl 2 were added sequentially. The samples were left for 15 min on ice and without the access of light.
• the criterion was the detection of at least 5,000 cells/sample.
• the actual detection of apoptosis in both methods was performed by flow cytometry (BD FACSVerse, BD Biosciences, San Jose, CA, USA) and the obtained data were analyzed using BD FACSuite software (BD Biosciences).
• Example 4 Detection of apoptosis and cell cycle phases in co-cultivated myeloma cells and fibroblasts The effects of complex 6 on the induction of apoptosis in two cell types (myeloma cells and fibroblast cells) co-cultured in the same medium were investigated.

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