Biological activity of semicarbazones and thiosemicarbazones

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NAMI-A, KP1019 and their analogues – ruthenium antimetastatic agents

In 1986, Bernhard Keppler described a new ruthenium(III) complex (imidazolium trans-[tetrachloride-bis(1H-imidazole)ruthenate(III)], KP418) which is consider as an inspiration of so-called « Keppler type » ruthenium complexes. This complex was proved therapeutic activity against P388 leukaemia, B16 melanoma and SA-1 (murine fibrosarcoma cells) cells lines[42, 43]. Moreover in vivo studies have shown a significant reduction of tumor burden in a colorectal cancer rats model[42]. Among all ruthenium(III) complexes synthesized on base of KP418, three are considered to be most important.
Imidazolium trans-[tetrachloride(1H-imidazole)(S-dimethylsulfoxide)ruthenate(III)] (NAMI-A) was firstly synthesized and studied in 1992 by Sava and co-workers[44]. In opposite to KP418, NAMI-A has much higher Ru(III/II) reduction potential due to -acceptor e ect of the S-bound dmso which also exerts a kinetic trans e ect. NAMI-A was proved to a ect the process of metastasing rather than acting against primary tumors. Its activity is probably based on enhanced cell adhesion and inhibition of neoangiogenesis in tumors[45, 46]. It was also proven that its mechanism of action does not involve DNA binding and its activity is rather independent of its concentration in cancer cells[37, 47, 48].
The other two significant Keppler complexes – KP1019 and NKP-1339 (respectively indazolium and sodium trans-[tetrachloridebis(1H-indazole) ruthenate(III)]) were described in 1989[49]. KP1019 was found to be superior in its activity against a colorectal cancer rats model then KP418. Treatment with KP1019 yielded e ciency with around 95% reduction of tumor volume with mortality at 0%. It was also found to be superior to 5-fluorouracil – a standard against colorectal cancer[43, 49]. Its activity comes from interactions between complex and DNA and inhibition of DNA synthesis[50].
One of the disadvantage of KP1019 is its solubility in water which may hinder the transfer in bloodstream. It also limit the administrative dose in clinical trials at maximum 600 mg/patient[51]. Its sodium salt (NKP-1339) on the other hand is much more water soluble. NKP-1339 exhibit similar activity as KP1019 in both in vitro and in vivo studies. It was determined that both compounds interact with serum proteins particularly albumin and transferrin similary to NAMI-A[52, 53]. It has been suggested that both proteins act as transport and delivery system for those complexes and play significant role in their cancer targeting mechanism.

Ruthenium complexes bearing biologically active molecules – theranostic application

As was mentioned in previous chapter, photophysical properties of ruthenium(II) polypyridyl complexes make them good candidates for luminescence probes. Recent studies have shown that the luminescence of those complexes is increased under the hypoxic conditions. However, the hydrophilic character of the complexes prevents them from crossing biological membranes and limits their applications. A series of novel ruthenium(II) complexes with better hydrophobic properties were synthesized few years ago. The complexes were composed of two 2,2’-bipyridine molecules and one 1,10-phenantroline bearing a long alkane chain or pyren unit. It was resulting in increased lipophilicity of the complex and better cells accumulation. Moreover, the luminescence properties of the complex were not a ected by presence of the moieties attached to the 1,10-phenantroline ligand[78].
The ruthenium(II) complex bearing the estradiol was designed as a specific probe for estrogen receptor . This receptor respond is the most accurate indicator of cancer in particular, estrogen-dependent cancer such as breast, colon, ovarian and prostate[79]. The other example of specific probes are ruthenium polypyridyl complexes bearing cumarin moiety. Those molecules were designed as esterase-specific sensors to measure the activity of the enzyme[80]. In order to target the nuclear enzymes, the ruthenium complexes were modified with the short peptides chains. The specific sequence of peptide can be recognize by specific enzyme. In this way, the complex can be modulate as a probe for only one enzyme.[71, 81].
However, the modulation of pharmacokinetic profile is not the only reason for introduction of additional groups into ruthenium complex structure. Recently, theranostic application of ruthenium complexes are extensively explored by many research groups. This novel approach is based on combining of therapeutic and diagnostic properties into one molecule. This method not only allows to monitor the therapeutic e ect of the treatment in real time, but also helps to visualize the biodistribution and accumulation of the drug.
In the most cases, conjugates of the ruthenium are received by binding the biologically active molecules with the complexes via linker. In general, the linker is a short, unbranched chain which can contain heteroatoms in its structure. The linker structures could have an important influence on properties of the ruthenium complexes. It was confirmed that structure of the linker have an influence on the cellular uptake and subcellular localization of the complex[82]. The complexes with the most hydrophilic linkers have the lowest cellular uptake and are mostly present in cytoplasm, while the complexes with the most hydrophobic linkers are localized mostly in mitochondria.
The ruthenium complex bearing 2-nitroimidazole was introduced recently as a new conjugate for treatment and visualization of cancer[83]. This molecule binds polypyridyl ruthenium(II) complex with the bioreductive prodrug, developed towards application in therapy of hypoxic cells, characteristic for tumors. The results have shown that this complex posses not only cytotoxic activity but also antiproliferative activity[84]. Ruthenium complexes were also adapted as luminescence probes[85] and photosensitizers in photodynamic therapy. A series of ruthenium complexes bearing porphyrins were designed as potential two-photon tumor-imaging and photodynamic agents[82, 86, 87]. The complexes had higher solubility and absorption band in the range between 600-800 nm – so called « therapeutic window » more appropriate for PDT – than porphyrins.
The theranostic concept was originally applied to much larger systems such as nanoparticles. Recent studies have shown that gadolinium based nanoparticles modified with ruthenium(II) phenantroline complexes can be used as imaging agents in MRI and simultaneously as photodynamic therapy agents[88]. Also binding ruthenium(II) bipyridine homoleptic complex with silver nanoparticles leads to drastically increased photoinduced oxidation activity[89].

Biological activity of semicarbazones and thiosemicarbazones

Semicarbazones and thiosemicarbazones are formed when semicarbazide – an ammonia related nucleofile – is added to carbonyl group (aldehyde or keton) and imine bond is formed. Historically, semicarbazones were used in qualitative analysis of aldehydes and ketons[90]. However, their wide spectrum of biological activity is known for more than 50 years. Many semicarbazones and thiosemicarbazones were evaluated as antibacterial, antiviral, antifungal, antifilarial and anticancer agents[91]. Properties and activity of those molecules strongly depends on substituent groups attached to both nitrogen atoms (N1 and N4) of semicarbazon or thiosemicarbazone. Also coordination to metal center plays important role in their activity.
The activity of some semicarbazones and thiosemicarbazones against Plasmodium parasites makes them a good candidates as an antimalarial drug mostly due to their inhibition of cysteine proteases[92]. They were also tested as anticonvulsant drugs due to their lesser neurotoxicity then other drugs[93, 94].

Ribonucleotide reductase – potential target in cancer treatment

Ribonucleotide reductase is an enzyme which is responsible for reduction of ribonucleotides into corresponding deoxyribonucleotides. Ribonucleotides reduction is a limiting step in DNA synthesis and hence inhibition of this step leads to inhibit cells proliferation. RNR exist in almost all living organisms – from bacteria to eukaryotes. It was even found in some viruses like bacteriophage or in archaea. It means that catalytic cycle of this enzyme is one of the oldest in the life cycle.
There are three main classes of ribonucleotide reductase which were discovered[98]. They di er in structure, the metal site and place of radical. Moreover class II and III can operate in anaerobic conditions while class I require oxygen. Main di erences were collected in table 1.2. Class I is most widely distributed in all eukaryotes. Presence of class II and III in eukaryotes is marginal (see table 1.1). Class I is further divided into two subclasses: Ia and Ib. Subclass Ib is mostly present in bacteria and Ia subclass is an enzyme present in eukaryotes’ cells[99].

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Hydantoins in cancer treatment

Besides antiepileptic properties of hydantoins, many of them were studied as potential anticancer agents[109, 110]. In fact, biological activity of hydantoins strongly depends on substituents attached to the hydantoin ring[109]. In general, hydantoins molecules can be divided into two parts[111]: N3-C4(=O)-C5-R which is consider as anticonvulsant pharmacophore and C2(=O)-N3(H) part which is mostly responsible for cytotoxic and mutagenic properties of the molecule. It is postulated, that modification of second part do not a ect anticonvulsant properties of the molecules but may result in changing toxicity and mutagenic properties of it.
Many new hydantoins derivatives had confirmed anticancer activity over last few years[110, 112, 113]. In major group of molecules only substituents at position 5 were changed, however also modification of N1 and N3 subtituents took place and even exchange of oxygen to sulfur[114] or selenium[113]. Results of this evaluation shown that some of the hydantoins posses high cytotoxicity towards cancer cells. They also are good antioxidants and antimetastatic agents.

Geometry optimalization and molecular orbitals energy calculations

The hybrid density functional B3LYP (Becke-Lee-Young-Parr composite of Exchange-correlation functional) was used for geometry and orbital energy calculation of studied ruthenium(II) complexes. For all complexes LANL2DZ basis-set [116, 117] was used for ruthenium atom and 6-31G(d,p) basis set for other atoms. The initial structure of ruthenium complex was based on crystallographic data of tris(2,2’-bipyridyl)dichlororuthenium(II)hexahydrate complex[118]. All calculations were performed with Gaussian 09 program[119] using PLGrid infrastructure. Orbitals were visualized using Gabedit software[120]. Structure figures and geometry parameters were obtained with Avogadro 1.1.1 software[121].

Electronic absorption spectra simulation

Electronic absorption spectra were simulated with time-dependent density functional theory (TD-DFT). Number of excited states to be determined was set to 40.The hybrid density functional B3LYP (Becke-Lee-Young-Parr composite of Exchange-correlation functional) was used with 3-21G basis-set for ruthenium atom and 6-31G(d,p) basis set for other atoms. The spectra were visualised with Gabedit software[120].

Spectrofluorimetric titration of human serum albumin with ruthenium complex

The solution of protein was prepared by dissolving of human serum albumin (HSA) in water and its concentration was determined spectrophotometrically from the molar absorbance coe cient of 4.40 104 M1 cm1 at 280 nm [124]. The emission spectra were recorded upon excitation at 295 nm resulting in selective excitation of Tryphtophan 214 residue. Three scans were subjected to smooth spectra and fluorescence intensity was corrected due to inner filter e ect according to equation 3.4. Fc = F0 10 ( Aex+Aem ) (3.4).
where F0 is recorded fluorescence intensity, Fc is corrected fluorescence intensity, Aex is absorbance of solution at excitation wavelength (295 nm) and Aem is absorbance of solution at emission wavelength (355 nm).
Protein binding experiments were performed by measuring fluorescence intensity of HSA solution (concentration 1 M) in the absence and the presence of various ruthenium complex concentration (up to 5 M) in TRIS-HCl bu er pH 7.4, concentration 0.1 M at 37°C and concentration of NaCl 0.1 M. Ru-protein solution was incubated for 10 minutes befor measurement.

Synthesis of ruthenium(II) complexes with ligands L2, L3 and L4

Similar procedure with few changes was applied to synthesize ruthenium(II) complexes with ligands L2, L3 and L4. For those ligands only one complex was used as the starting material – cis-bis(2,2’-bipyridine)dichlororuthenium(II). The complex was dissolved in absolute ethanol and proper ligand was added. Mixture was refluxed under argon for 18 h (followed by TLC) and solvent was removed under reduced pressure. The residue was dissolved in small amount of acetonitrile and saturated solution of potassium hexafluorophosphate was added in large excess. The orange solid was filtrated, washed with cold water and dried. The pure product was obtained after flash chromatography (neutral Al2O3, dichloromethane/methanol 9/1). Synthesis of those complexes is presented in the figure 4.17.

Table of contents :

1. Introduction
1.1. History of medicinal inorganic chemistry
1.1.1. Metals in medicine
1.1.2. Cisplatin – the first anticancer drug
1.1.3. Reducing side-eects – Cisplatin analogues
1.2. Ruthenium complexes in cancer treatment
1.2.1. Simple ammine and amine ruthenium complexes
1.2.2. Ruthenium dimethyl sulfoxide complexes
1.2.3. NAMI-A, KP1019 and their analogues – ruthenium antimetastatic agents
1.2.4. Ruthenium-arene complexes
1.2.5. Ruthenium polypyridyl complexes
1.2.6. Ruthenium complexes bearing biologically active molecules – theranostic application
1.3. Biological activity of semicarbazones and thiosemicarbazones
1.3.1. Anticancer activity
1.3.2. Ribonucleotide reductase – potential target in cancer treatment
1.4. Hydantoins – anticonvulsant drugs in cancer treatment
1.4.1. Seizures and cancer
1.4.2. Hydantoins in cancer treatment
2. Aim and scope of the thesis
3. Methods
3.1. Electronic apsorption spectroscopy
3.2. Luminescence studies
3.3. Computational studies
3.3.1. Geometry optimalization and molecular orbitals energy calculations
3.3.2. Electronic absorption spectra simulation
3.4. Synthesis
3.5. Spectrofluorimetric titration of human serum albumin with ruthenium complex
4. Results and discussion
4.1. General strategy of ruthenium(II) polypyridyl complexes synthesis
4.2. Synthesis of 2,2’-bipyridine ligands modified with pyridine-2-carboxyaldehyde semicarbazone and the linker – various approaches Synthesis of ligand L1 – 5-(4-{4’-methyl-[2,2’-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone
4.3. Synthesis of 3-(5-{4’-methyl-[2,2’-bipyridine]-4-yl}pentyl) -imidazolidine-2,4-dione (L2),
5,5-dimethyl-3-(5-{4’-methyl-[2,2’-bipyridine]-4-yl}pentyl) -imidazolidine-2,4-dione (L3) and [1-(5-{4’-methyl-[2,2’-bipyridine]-4-yl}pentyl) -2,5-dioxoimidazolidin-4-yl]urea (L4) – General procedure
4.4. Ruthenium complexes synthesis
4.4.1. Synthesis of cis-bis(polypyridine)dichlororuthenium(II)
4.4.2. Synthesis of ruthenium(II) complexes with ligand L1
4.4.3. Synthesis of ruthenium(II) complexes with ligands L2, L3 and L4
4.5. Photophysical properties of ruthenium complexes
4.5.1. Absorption properties
4.5.2. Emission properties
4.6. Computational studies
4.6.1. Geometry calculations
4.6.2. Natural Bond Orbitals (NBO) analysis
4.6.3. Electronic absorption spectra
4.7. Ruthenium complexes intractions with Human Serum Albumin
4.7.1. Human Serum Albumin properties
4.7.2. Interactions with ruthenium complexes
5. Summary
5.1. Preliminary cytotoxic studies – Future perspectives
6. Synthetic procedures
6.1. Synthesis of Ligand L1
6.1.1. Synthesis of 4-(3-(trimethylsilyl)prop-2-yn-1-yl)-4’-methyl-2,2’-bipyridine (1)
6.1.2. Synthesis of 4-(prop-2-yn-1-yl)-4’-methyl-2,2’-bipyridine (2)
6.1.3. Synthesis of 5-bromopyridine-2-carboxyaldehyde (3)
6.1.4. Synthesis of 5-bromopyridine-2-carboxyaldehyde semicarbazone (4)
6.1.5. Synthesis of 5-(4-{4’-methyl-[2,2’-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone (L1)
6.2. Synthesis of Ligand L2, L3 and L4
6.2.1. Synthesis of 4-(5-bromopentyl)-4’-methyl-2,2’-bipyridine (6)
6.2.2. Synthesis of 3-(5-{4’-methyl-[2,2’-bipyridine]-4-yl}pentyl)- -imidazolidine-2,4-dione (L2)
6.2.3. Synthesis of 5,5-dimethyl-3-(5-{4’-methyl-[2,2’-bipyridine]-4-yl}pentyl)- -imidazolidine-2,4-dione (L3)
6.2.4. Synthesis of [1-(5-{4’-methyl-[2,2’-bipyridine]-4-yl}pentyl)- -2,5-dioxoimidazolidin-4-yl]urea (L4)
6.3. Ruthenium complexes synthesis
6.3.1. General procedure for synthesis of cis-Ru(NN)2Cl2
6.3.2. Synthesis of [Ru(bpy)2(L1)]Cl2 (16)
6.3.3. Synthesis of [Ru(Mebpy)2(L1)]Cl2 (17)
6.3.4. Synthesis of [Ru(tBbpy)2(L1)]Cl2 (18)
6.3.5. Synthesis of [Ru(Phbpy)2(L1)]Cl2 (19)
6.3.6. Synthesis of [Ru(dip)2(L1)]Cl2 (20)
6.3.7. Synthesis of [Ru(SO3dip)2(L1)]Cl2 (21)
6.3.8. Synthesis of [Ru(bpy)2(L2)](PF6)2 (22)
6.3.9. Synthesis of [Ru(bpy)2(L3)](PF6)2 (23)
6.3.10. Synthesis of [Ru(bpy)2(L4)](PF6)2 (24)
6.4. Other synthesis
6.4.1. Synthesis of 2-bromo-4-methylpyridine (25)
6.4.2. Synthesis of 4-methylpyridine-2-carboxyaldehyde (26)
6.4.3. Synthesis of 2-(1,3)-dioxolan-2-yl-4-methylpyridine (27)
6.4.4. Synthesis of 4-(7-bromoheptyl)-4’-methyl-2,2’-bipyridine (28)
6.4.5. Synthesis of 2-bromo-4-[6-(2-bromopyridin-4-yl)hexyl]pyridine (29)
6.4.6. Synthesis of 2-bromopyridine-5-carboxyaldehyde (30)
6.4.7. Synthesis of (2-bromopyridin-5-yl)methanol (31)
6.4.8. Synthesis of ({4’-methyl-[2,2’-bipyridine]-4-yl}pentyl)triphenylphosphonium bromide (32)
6.4.9. Synthesis of diethyl ({4’-methyl-[2,2’-bipyridine]-4-yl}pentyl)phosphonate (33)


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