Classifications

• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the invention that belongs to the area of the platinum-based drugs against tumour diseases, relates to the platinum cyclobutane-1 , 1 -dicarboxylato complexes involving the N6-benzyladenine derivatives, and their use in the medical practice as drugs and pharmaceutical compositions, which contain these complexes as the active substance.
• Cisplatin (a), c/ ' s-[PtCI 2 (NH 3 ) 2 ], c/ ' s-diamminedichlorido-platinum(ll) complex is one of the well-known platinum complexes used for the treatment of cancer (Rosenberg, B.; Camp, V.; Krigas, T. Nature 1965, 205, 689-699). It has been used in chemotherapy against tumours of testis, ovary, neck, head, or other types of tumour diseases (Weiss, R. B.; Christian, M. C. Drugs 1993, 46, 360-365). The chemotherapy itself is accompanied by negative side effects on the organism, such as nephrotoxicity or ototoxicity.
• Carboplatin (b) is effective against almost the same human cancer cell lines as cisplatin (a) and it is also administered intravenously (Hambley, T. W.; Dalton Trans. 2001, 120, 2711-2718). Contrary to cisplatin the toxicity to organism is much lower, which leads to the significant decrease of the negative side effects ( Wilkinson, A. R.; Cox, P. J.; Jones, M.; Harrap, K. R. Biochimie 1978, 60, 851). Carboplatin (called JM- 8) was the first substance to be reported as in vivo cytotoxic active against testicular cancer, lung cancer (V79) and breast tumour (MTG-B) in 1985 (Peckham, M.
• oxaliplatin [ 7R,2/R-diamminecyclohexane-N,N ' -]-(oxalato-0,0 ' )platinum(ll) complex, synthesized for the first time in 1978 (Kidani, Y.; Inagaki, K. J. Med. Chem., 1978, 21, 1315- 1318, EP2029615 - A proces for the preparation of an oxaliplatin, EP1979369 - Preparation of platinum(ll) complexes), whose molecular structure was determined in 1984 (Bruck, M.
• N6-benzyladenine itself belongs to the group of natural plant growth regulators called cytokinins (Skoog, F.; Hamzi, H. Q.; Szweykowska, A. M.; Leopard, N. J.; Carraway, K. L; Fujii, T.; Helgeson, J. P.; Loeppky, R. N. Phytochem. 1967, 6, 1169-1192).
• WO 0043394 Substituted nitrogen heterocyclic derivatives process of their preparation, the derivatives employed as medicaments, pharmaceutical composition and a compound pharmaceutical preparation in which these derivatives are comprised as well as use of these derivatives for preparing medicaments
• WO 03040144 Heterocyclic compounds based on N6-substituted adenine, processes of their preparation, their use for preparing medicaments, cosmetic compositions and growth regulators and pharmaceutical preparations, cosmetic compositions and growth regulators containing such compounds
• WO 0149688 Purine derivatives, process of their preparation and use
• WO 2004058791 Substituting derivatives of N6-benzyladenosine, process of their preparation, their use in the
• L 8 4,4-bis- (aminomethyl)tetrahydrofuran (Bitha, P.; Child, R. G.; Hlavka, J. J.; Lang, S.A.; Lin, Y - /.; Haltiwanger, R. C.; Pierpont, C. G. Inorg. Chim. Acta 1988, 151, 89-93),
• L 9 1 ,3- dihydroxy-2,2-bis(aminomethyl)propane (Bitha, P.; Child, R. G.; Hlavka, J.J.; Lang, S.A.; Lin, Y.-i; Haltiwanger, R. C.; Pierpont, C.
• L 12 bis(2-pyridyl)ketone (Ferreira, A.D. Q.; Bino, A.; Gibbon, D. Inorg. Chim. Acta 1997, 265, 155-161)
• L 13 (3,6,9, 12- tetraoxatetradecane-1 , 14-diyl-2,2'-bipyridyl-3,3'-dicarboxylate (Yoo, J.; Sohn, Y.S.; Do, Y. J. Inorg. Biochem. 1999, 73, 187-193)
• L 14 2,3-bis-(2-pyridyl)pyrazine (Ferreira, A.D.
• Cyclobutane-1 , 1-dicarboxylato complexes of platinum in the oxidation state +II and their crystal-solvates including a structural motif I or having the general formula II expressed by the structural formula [Pt(cbdc)(L) 2 ] or the general formula III expressed by the structural formula [Pt(cbdc)(L)(L ' )], where the symbols L and L ' stand for N6-benzyladenine derivatives of the general formula IV bonded to the platinum atom of the basic motif V through any adenine nitrogen atom independently chosen from the N1 , N3, N6, N7 or N9 atoms, depending on the substitution rate of the molecules, where the substituent R1 , R2, R3 and R4 are independently chosen from the group of:
• - alkenyl as employed herein by itself or as a part of another group represents a straight or branched hydrocarbon chain from up to 18, preferably from 2 to 8 carbon atoms, including at least one carbon-carbon double bond,
• cyclic hydrocarbon chain from 3 to 12, preferably from 3 to 8 carbon atoms, which may be fused with 1 or 2 cycles which are independently selected from the group consisting of cycloalkyl, cycloheteroalkyl, cycloalkenyl, cycloheteroalkenyl, aryl a heteroaryl,
• cykloalkyl where at least one carbon atom is exchanged for a heteroatom from the group consisting of nitrogen, oxygen, sulphur,
• cycloheteroalkyl including at least one carbon-carbon, carbon-heteroatom, or heteroatom-heteroatom double bond
• - functional group as employed herein by itself or as a part of another group represents amino, al- kylamino, dialkylamino, alkenylamino, cycloalkylamino, cycloheteroalkylamino, cyc- loalkenylamino, cycloheteroalkenylamino, arylamino, heteroarylamino, acylamino, hydroxy, alkylhydroxy, alkoxy, aryloxy, cyano, carboxy, alkylcarboxy, carboxyalkyl, aryl- carboxy, carboxyaryl, hydroxyamino, acyl, nitro, amido, nitroso, sulfonyl, sulfinyl, sulf- amido, thio, alkylthio, arylthio, merkapto, carbamoyl, - - substituted alkyl, substituted alkenyl, substituted alkynyl, substitute
• alkyl alkenyl, alkynyl, cycloalkyl, cycloheteroalkyl, cycloalkenyl, cycloheteroalkenyl, aryl and heteroaryl substituted by the substituents from the group consisting of halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloheteroalkyl, cycloalkenyl, cycloheteroalkenyl, aryl, heteroaryl and a functional group.
• the next objective of the invention are crystal-solvates of the platinum(ll) cyclobu- tane-1 , 1 -dicarboxylato complex of the general formula [Pt(cbdc)(L) 2 ] xSolv or [Pt(cbdc) (L)(L ' )] xSolv, where (xj represents the number of the solvate molecules, preferably 1 to 6, and (Solv) represents a concrete molecule of the used solvent and/or some of the reaction components, usually chosen from the group consisting of water, primary alcohol, secondary alcohol, acetone, ⁇ /, ⁇ / ' -dimethylformamide (DMF), dimethyl sulfoxide (DMSO), chloroform, dichloromethane, acetonitrile or diethyl ether, either individually or in combinations of the mentioned solvate molecules.
• DMF dimethyl sulfoxide
• the invention also provides a pharmaceutical and pharmacological substance containing a therapeutically effective amount of the platinum(ll) cyclobutane-1 , 1 -di- carboxylato complexes or a pharmaceutical composition of the platinum(ll) cyclobutane-1 , 1 -dicarboxylato complexes with one or more acceptable carriers and additional substances for medicinal use as a drug for tumour diseases.
• Fig. 1 represents the 13 C NMR spectrum of bis[2-chloro-6-(3,4-dichlorobenzyl)- amino-9-isopropylpurino]-cyclobutane-1 , 1 -dicarboxylato-platinum(ll) complex, [Pt(cbdc)(L 2 )2], given with the interpretation of the individual carbon atoms, which were detected by the 13 C, APT, 1 H- 13 C gs-HMQC and 1 H- 13 C gs-HMBC experiments
• Fig. 2 represents the FT IR spectrum of the bis[2-chloro-6-(2-fluoro-5-bromobenzyl)- amino-9-isopropylpurino]-cyclobutane-1 ,1-dicarboxylato-platinum(ll) complex, [Pt(cbdc)(L ) 2 ], measured in the 400-4000 cm "1 range and given with the maxima characterizing the individual atom groups
• Fig. 3 represents the FT IR spectrum of the bis[2-chloro-6-(2-fluoro-5-bromobenzyl)- amino-9-isopropylpurino]-cyclobutane-1 , 1 -dicarboxylato-platinum(ll) complex, [Pt(cbdc)(L 8 ) 2 ], measured in the 150-4000 cm -1 range and given with the maxima characterizing the Pt-N and Pt-0 vibrations
• Fig. 4 represents the Raman spectrum of the bis[2-(3-hydroxypropylamino)-6- benzylamino-9-isopropylpurino]-cyclobutane-1 ,1-dicarboxylato-platinum(ll) complex, [Pt(cbdc)(L 1 ) 2 ], measured in the 400-4000 cm "1 range and given with the maxima characterizing the individual atom groups
• Fig. 5 represents the molecular structure of the complex [2-chloro-6-(2- methoxybenzyl)amino-9- ' isopropylpurino]-(dimethylsulfoxid-S)-cyclobutane-1 ,1-dicarb- oxylato-platinum(ll) complex monohydrate, [Pt(cbdc)(DMSO)(L 7 )] H 2 0, determined by a single crystal X-ray analysis and with non-hydrogen atoms drawn as thermal ellipsoids at the 50% probability level. H-atoms are omitted for clarity.
• the derivative L ' can be, from the chemical and structural point of view, towards L:
• the invention also uses c/s-[PtCI 2 (DMSO) 2 ] and [Pt(cbdc)(DMSO) 2 ], where cbdc is dianion of the cyclobutane- ,1 -dicarboxylic acid, as the intermediates for the preparation of platinum cyclobutane-1 , 1 -dicarboxylato complexes of the general formula II or III, which can be prepared by the reaction of platinum chloride, PtCI 2 , with dimethyl sulfoxide (DMSO) leading, at raised temperature, to c/ ' s-[PtCI 2 (DMSO) 2 ] (Rochon, F. D.; Massarweh, G. Inorg. Chim.
• Scheme 1 salt is added.
• the reaction mixture is stirred in darkness at low temperature for several days.
• the [Pt(cbdc)(DMSO) 2 ] complex is formed (Scheme 1 ).
• Detail description of the preparation of this intermediate was reported in Bitha, P.; Morton, G. O.; Dunne, T. S.; Santos, E. F. D.; Lin, Y.; Boone, S. R.; Haltiwanger, R. C; Pierpoint, C. G. Inorg. Chem. 1990, 29, 645-652.
• FT IR infrared spectroscopy
• the invention employs [Pt(cbdc)(DMSO) 2 ] as a starting platinum compound.
• This complex is not commercially available, but it can be prepared according to the synthetic procedure described in the literature (Bitha, P.; Morton, G. O.; Dunne, T. S.; Santos, E. F. D.; Lin, Y.; Boone, S. R.; Haltiwanger, R. C; Pierpoint, C. G. Inorg. Chem. 1990, 29, 645-652, Scheme 2).
• the percentual content of the C, H, N elements given as: calculated value (found value in parenthesis) for CioHi 8 0 6 PtS 2 : C, 24.3 (23.9); H, 3.7 (3.9)%.
• Raman (i/KBr/crrr 1 ): 261 (w, Pt-N), 479 (w, Pt-O), 890 (m, C-H), 1339 (vs, N- H), 1586 (s, C N), 2942 (s, C-H 2 ), 3064 (s, C-H).
• Example 2 Synthesis of bis[2-chloro-6-(2-fluoro-5-bromobenzylamino)-9-isopropyl- purino]-(cyclobutan-1 ,1-dicarboxylato)-platinum(ll) complex, [Pt(cbdc)(Li) 2 ] 0.1 g (0.2 mmol) of [Pt(cbdc)(DMSO) 2 ] was dissolved in 15 mL of distilled water at laboratory temperature. The solution of 0.16 g (0.4 mmol) of 2-chloro-6-(2-fluoro-6- bromobenzyl)amino-9-isopropylpurine (L ⁇ in 20 mL of isopropanol was added.
• reaction mixture was stirred for 2 days until the grey product formed. It was filtered off, washed with cold distilled water and dried in dissicator over silica gel (Scheme 4).
• the obtained complex was characterized by elemental analysis, FT IR, Raman and 1 H, 3 C and 195 Pt NMR spectroscopy.
• Example 3 Synthesis of bis[2-(3-hydroxypropylamino)-6-benzylamino-9-isopropylpu- rino]-(cyclobutan-1 ,1-dicarboxylato)-platinum(ll) complex, [Pt(cbdc)(L 8 ) 2 ] 0.1 g (0.2 mmol) of [Pt(cbdc)(DMSO) 2 ] was dissolved in 15 mL of distilled water at laboratory temperature. The solution of 0.136 g (0.4 mmol) of 2-(3- hydroxypropylamino)-6-benzylamino-9-isopropylpurine (L 8 ) in 20 mL of isopropanol was added.
• reaction mixture was stirred for 2 days until the grey product formed. It was filtered off, washed with cold distilled water and dried in dissicator over silica gel (Scheme 5).
• the obtained complex was characterized by elemental analysis, FT IR, Raman and 1 H, 13 C and 95 Pt NMR spectroscopy.
• Example 4 Synthesis of dimethylsulfoxido-[2-chloro-6-(2-methoxybenzylamino)-9- isopropylpurino]-(cyclobutan-1 ,1-dicarboxylato)-platinum(ll) complex, [Pt(cbdc) (DMSO)(L 7 )] 0.1 g (0.2 mmol) of [Pt(cbdc)(DMSO) 2 ] was dissolved in 15 mL of distilled water at laboratory temperature. The solution of 0.08 g (0.2 mmol) of 2-chloro-6-(2- methoxybenzyl)amino-9-isopropylpurine (L?) in 20 ml_ of isopropanol was added. The reaction mixture was stirred for 2 days (Scheme 6). The obtained grey product was filtered off, washed with cold distilled water and dried in dissicator over silica gel.
• the colourless crystals suitable for a single crystal X-ray analysis, formed in the filtrate in several days.
• the molecular structure of [Pt(cbdc)(DMSO)(L 7 )] H 2 0 was determined by the mentioned method using Oxford Xcalibur diffractrometer equipped by CrysAlis CCD detector (Oxford Diffraction, 2002). Data collection and reduction were performed using CrysAlis software [CrysAlis CCD and CrysAlis RED, Version 1.171.33.52, Oxford Diffraction Ltd., Abingdon, England, 2009]. The same software was used for data correction for an absorption effect by the empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm.
• One of the parameters, used for the evaluation of in vitro cytotoxic activity, is metabolic activity of viable cells.
• micro-titration testing employing a Calcein AM is widely used for the quantification of cell proliferation and cytotoxicity. This method is applied for the screening of drugs or for chemosensitivity testing. This test recognizes only living cells and the degree of Calcein AM reduction corresponds to the amount of living cells.
• a screening of in vitro cytotoxicity was performed against the breast adenocarcinoma (MCF-7) and chronic myelogenous erythroleukemia (K562) human cancer cell lines.
• the cells were maintained in plastic tissue culture flasks in the DMEM medium (5 g/L of glucose, 2 mM of glutamine, 100 U/mL of penicillin, 100 pg/mL of streptomycine, 10% fetal calf serum and sodium hydrogen carbonate) for cell cultures.
• the cell suspensions (ca 1.25 x 10 5 cells mL ) were pipetted (80 ⁇ _) into 96-well microplates. They were incubated at 37 °C in the C0 2 atmosphere for 24 hrs.
• the tested platinum(ll) complexes were dissolved in N,N -dimethylformamide, diluted to the 0.2-25.0 ⁇ concentration and added to the incubated cancer cell suspension. Then the mixtures were incubated for 72 hrs at 37 °C, 100% humidity and in the C0 2 atmosphere. The Calcein AM solution was added followed by the incubation lasting 1 hr.
• the living cancer cell fluorescence (FD) was measured at 485/538 nm (excitation/emission) on the Labsystem FIA Fluoroscan Ascent device (Microsystems).
• the IC 50 values represent the concentration of the tested complexes lethal for 50% of the cancer cells.
• the method is based on the ability of metabolic-active cells to reduce (by mitochondrial dehydrogenases) the yellow salt 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) leading to the blue formazane dye formation. Because the reduction proceeds only in the living cells, the cytotoxicity of various compounds can be determined using this process.
• the insoluble formazane is quantified after dissolving in dimethyl sulfoxide (DMSO) with ammonium on the spectrophotometer (ELISA reader).
• DMSO dimethyl sulfoxide
• ELISA reader spectrophotometer

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