BRPI1002523A2 - synthesis process of copper complexes with antitumor activity - Google Patents

Abstract

SYNTHESIS PROCESS OF COPPER COMPLEXES WITH ANTITUMORAL ACTIVITY. The present invention describes the synthesis and characterization of ternary copper complexes having as ligands the 1.10-phenanthroline or its derivatives and antibiotics of the family of tetracyclines, chemically modified or not, or ligands of the group of the hydrazides substituted of the furic acid or benzoic acid, as well as pharmaceutical compositions containing them, as antineoplastic agents. The innovative aspect of this invention includes both the described complexes, which are new, the method of preparing them, as well as their anti-tumor activities.

Description

"SUMMARY PROCESS OF COPPER COMPLEX WITH ANTITUMORAL ACTIVITY"

Field of the invention

The present invention describes the synthesis and characterization of copper ternary complexes having as binders 1,10-phenanthroline or its derivatives and chemically modified tetracycline family antibiotics, or linkers of the substituted folic acid or hydrazide group benzoic acid as well as pharmaceutical compositions containing them as antineoplastic agents.

The innovative aspect of this invention includes both the described complexes, which are unprecedented, the method of preparation thereof, as well as their anti-tumor activities.

State of the art

Research into metal complexes with antitumor properties began with the discovery of the antitumor action of cisplatin, cis-diaminodichloroplatin (ll), by Rosenberg and Van Camp in the late 1960s (B. Rosenberg, L. Van Camp, JE Trosco, VH Mansour, Nature, 222,1969, 385) and has been stimulated with the discovery of cytotoxic activity of complexes with other metals such as copper, ruthenium, gallium, vanadium, palladium, titanium, etc. (CX Zhang, SJ Lippard, Curr. Opin, Chem, Biol., 7,2003,481). The introduction of cisplatin in the medical clinic in 1971 undoubtedly had a major impact, enabling complete healing of several tumors, particularly the testicular and ovarian tumors (B. Rosenberg, L. Van Camp, Cancer Research, 30, 1970, 1799). Unfortunately, despite cisplatin's therapeutic success, it has some disadvantages such as low water solubility, undesirable side effects, and the appearance of cell resistance. This is followed by a search for compounds that exhibit better solubility, have fewer side effects and activity in cisplatin-resistant tumors.

DNA is the main target of antitumor drugs. Metal complexes can bind directly to DNA, such as cisplatin (AMJ Fichtinger-Schepman, JL Van der Veer, JHJ den Hartog, PHM Lohman, J. Reedijk, Biochemistry, 24, 1985, 707), or indirectly, through oxy-reduction reactions, generating active species that can break down the DNA1 molecule such as bleomycin, a natural and potent antitumor glycopeptide (J. Reedijk, Proc. Natl. Acad. Sci. USA, 100, 2003, 3611).

Some transition metal complexes can promote free radical formation via the Fenton reaction and Haber-Weiss reaction. Radical species that degrade the ribose ring of DNA are formed.

On the other hand, the 1,10-phenanthroline compound and its derivatives are of interest in the synthesis of novel antitumor activity metallopharmaceuticals, since they have flat structures and are therefore capable of interleaving the DNA strand and preventing replication. cell phone. Another advantage of these compounds is the lack of antibacterial activity against the intestinal flora, which reduces the side effects of a possible metallopharmaceutical with these compounds (S. Roy, KD Hagen, PU Maheswari, M. Lutz, Spek AL, J. Reedijk, GP van Wezel, Chem. Med. Chem., 3, 9, 2008, 1427).

A well-studied example is the 1,10-phenanthroline Cu (II) complex. The reactive species [Cu (Phen) 2] + makes an oxidative attack on a ribose carbon. Hydrogen peroxide may be generated by oxygen reduction by the species [Cu (Phen) 2] + (T. Hirohama, Y. Kuranuki, E. Ebina, T. Sugizaki, H. Arii, M. Chikira, PT Selvi, M. Palaniandavar, J. Inorg, Biochem., 99, 2005, 1205).

Some patents containing metal compounds with 1,10-phenanthroline derivatives that have antitumor activities are available in the literature. Examples include two US patents: 4,699,978 (Site-specific chiral ruthenium (II) and cobalt (III) antitumor agents) and 5,225,556 (Chemical probes for left-handed DNA and A-DNA; chiral metal complexes as Z-specific antitumor agents and as double strand cleavers) comprising syntheses, characterizations and cytotoxic studies of complexes with 1,10-phenanthroline derivatives. They have been shown to be effective in breaking down the DNA molecule and thereby preventing the proliferation of tumor cells. Two Chinese patent applications filed with the European Patent Office can also be cited: CN101190925 (Copper complex, preparation method and application) and CN101225092 (Manganese complex as well as preparation method and uses) which describe the synthesis, characterization and application of complexes. copper and manganese with 1,10-phenanthroline as antitumor agents.

On the other hand, tetracyclines are widely known for their antibiotic properties, having several advantages over currently known agents such as broad spectrum of action, low toxicity, oral administration and low cost. However, this family of compounds has other pharmacological properties besides antimicrobial activity. Some patents containing tetracyclines and analogues as compounds capable of inhibiting cancer growth are already available in the literature. Examples of two US patent applications are: WO / 1998/031224 (Method of inhibiting cancer growth) which comprises the use of a tetracycline analogue to induce cytotoxic effects against cancer in mammals and is particularly effective for inhibiting cancer. formation, growth and metastasis of solid tumors such as tumors derived from colon, breast, prostate or lung cancer cells and WO / 1987/003489 (Combinations of tumor necrosis factors and antibiotics and methods for treating tumors) refers to methods for treating tumors and neoplastic diseases with the combination of antitumor drugs and tetracycline in order to inhibit protein synthesis in the target cell.

The perspective of pharmaceutical use of tetracycline or metal complexed derivatives, too, is another strand of studies. US patent application WO0 / 2003/066064 (Methods of disease treatment using metal-complexed tetracycline antibiotics) describes the application of complexed tetracyclines and quinones compounds to metals, iron, copper and calcium for the treatment of bacterial diseases.

Doxycycline and other chemically modified tetracyclines have also been reported to inhibit matrix metalloproteinases, which are involved in different stages of cancer progression. This inhibition has been suggested to be related to the coordination to protein zinc or calcium atoms (RA Garcia, DP Pantazates, CR Gessner, KV Go, VL Woods Jr., FJ Villarreal, Mol. Pharmacoi 67, 4, 2005,1128) .

In addition, recent studies show that platinum (II) complexes with doxycycline and tetracycline inhibit chronic myeloid leukemia cell growth and this antitumor activity is possibly related to the interaction of the complex with DNA by intercalative mechanism (PP Silva, FCS de Paula , W. Guerra, JN Silveira, FV Botelho, LQ Vieira, T. Bortolotto, FL Fischer, G. Bussi, H. Terenzi, EC Pereira-Maia, J. Braz. Chem. Soc., 21, 2010,1237).

Another strategy that can be used for the development of anticancer drugs is to combine the antitumor properties of the compound [Cu (Phen) 2] 2+ with the hydrazide derivatives, since the latter also have neoplastic properties. On the antitumor activity of hydrazide derivatives, we can cite Brazilian patent application PI0512526-0 (Compound, pharmaceutical composition, method for treating a cancer patient and method for preparing a bis (thiohydrazide amide) di-salt which suggests the use of a pharmaceutical composition comprising a hydrazide-derived salt, bis (thiohydrazide amide) salt, for treating cancer patients.

Detailed Description of the Invention

The process of synthesis and characterization of new metallopharmaceuticals as anticancer agents is proposed in this invention, where the focused metal is copper, since it is an endogenous metal and possibly less toxic than platinum. The ligands employed may be tetracycline (tc), doxycycline (dox), minocycline (min), tigecycline (tig), all belonging to the tetracycline family, benzoic acid (hydb) or furic acid (hydf) hydrazides ), as well as 1,10-phenanthroline (phen) or derivatives thereof, 2,9-dimethyl-1,10-phenanthroline (neocuprine) (2,9-dmphen) or 4,7-diphenii-1,10 - phenanthroline (batophenanthroline) (4,7-dfphen). The present invention proposes the synthesis of novel compounds that can be used in pharmaceutical formulations to treat cases of cancer. The structures of three of the compounds [Cu (R) (R1) (R2) (ClO4) I (ClO4), where R = tc, dox, min, tig, hydf, hydb; R1 = phen, 2,9-dmphen, 4,7-dfphen and R2 = H 2 O, acetonitrile, are presented in Figure 1. The compounds were synthesized and characterized by elemental and conductivity analysis, electronic paramagnetic resonance (EPR), spectroscopy on the ultraviolet-visible (UV-Vis) and infrared (IR), mass spectrometry and single crystal X-ray crystallography for complex III.

All compounds exhibit significant cytotoxic activity in tumor cell lines and may be used to treat cancer.

The compositions of the present invention are characterized by the use of copper complexes combined with pharmaceutically acceptable excipients. Standard compositions may be liquid and solid. The liquid preparations may be in solution, suspension or emulsion form. Solids in the form of capsules, tablets, dragees or lozenges.

Such compositions may be administered intramuscularly, intravenously, orally or as devices that may be implanted or injected.

The technology can be better understood by looking at the following non-limiting examples:

A) Synthesis of complexes

Example 1 - Synthesis of complexes I and II:

Complex I was prepared by reacting Cu (ClO4) 2-6H20 (0.1853g, 0.5 mmol) with doxycycline (0.2405g, 0.5 mmol) in water (10 mL). The mixture was stirred for 40 minutes, then 5 mL of a 1,10-phenanthroline (0.0996g, 0.5 mmol) methanolic solution was added. The resulting mixture was stirred for a further three hours. The formed precipitate was filtered off, washed with water and dried under vacuum. A green solid of the composition [Cu (C 22 H 24 N 2 O 8) (C 12 H 8 N 2) (H 2 O) (ClO 4)] (ClO 4) was obtained. Complex II was similarly prepared with the addition of tetracycline instead of doxycycline.

Example 2 - Synthesis of complex III:

Complex III was prepared by reacting Cu (ClO4) 2-6H20 (0.1853g, 0.5 mmol) with 2-furic acid hydrazide (0.2405g, 0.5 mmol) in acetonitrile (5 mL) followed by addition of a solution of 1,10-phenanthroline (0.0996g, 0.5 mmol) in acetonitrile (3 mL). Blue crystals of the composition [Cu (C 5 H 6 N 2 O 2) (C 12 H 8 N 2) (C 2 H 3 N) (ClO 4)] (ClO 4) were obtained.

A similar complex can also be obtained using water instead of acetonitrile as a solvent, the final composition of which is [CU (C5H6N2O2) (C12H8N2) (H2O) (ClO4)] (ClO4).

B) Characterization of copper complexes with tetracyclines (complexes I and II) and with 2-furic acid hydrazide (complex III)

The complexes were obtained according to the following scheme:

Cursed) + L1 (SO1) CuL1 + L2 (SO1) - »· CI, CII or CIII

Being complex I (CI) = [CuL1 L2 (ClO4) (H2O)] ClO4, when sol1 = water and sol2 = methanol, L1 = dox and L2 = phen; complex II (Cu) = [CuL1 L2 (ClO4) (H20)] ClO4, when sol1 = water and sol2 = methanol, L1 = tc and L2 = phen and complex Ill (Cm) = [CuL1L2 (ClO4) (CH3CN )] ClO4, when sol1 = water and sol2 = acetonitrile, L1 = hydf and L2 = phen.

The compounds were characterized by elemental analysis, conductivity, EPR, IR and UV-Vis spectrometry, mass spectrometry (complex I and II) and single crystal X-ray crystallography (complex III). Elemental analysis and molar conductivity data are reported in Table 1 and RPE data are reported in Table 2. Table 1: Elemental and conductivity analysis data for complexes I, II and III. The values in parentheses are calculated for [Cu (C12H8N2) (C22H24N2O8) (H2O) (ClO4)] (ClO4), complexes I and II and [Cu (C5H6N202) (C12H8N2) (C2H3N) (ClO4)] (ClO4), complex III.

<table> table see original document page 8 </column> </row> <table>

The elemental analysis data are consistent with the proposed formulas. Molar conductivity was measured in nitromethane solutions (10 "3 molL" 1) at 25 ° C. The values obtained for these three complexes indicate that, in solution, they are type 2: 1 electrolytes. This means that, in solution, the two perchlorate ions act as counter ions.

Table 2: RPE parameters for complexes I, II and III.

<table> table see original document page 8 </column> </row> <table>

The values obtained are in accordance with the proposed structure and the EPR spectra of complexes I and III can be observed in Figure 2. The EPR spectrum of compound II is identical to that of compound I.

Analysis of UV-Vis absorption spectra for complexes I and II suggested the involvement of tc and dox ring A in metal coordination. The absorption band at 269 shifted to 293 nm in complex I as shown in Figure 3a and from 270 to 296 nm in complex II (Figure 3b), while the 362 nm centered band for tc BCD chromophore or dox did not change in both complexes. For complex III, the phen band centered at 264 nm undergoes a batochromic shift to 272 nm in the complex indicating the ligand-metal complexation (Figure 3c).

Absorption assignments in infrared spectra of complexes I and II were based on a detailed study of tetracyclines and analogues by Dziegielewski et al. (J. Dziegielewski, J. Hanuza, B. Jezowska-Trzebiatowska, Bull Acad. PoL ScL, Ser. Sci. Chim. 24, 1976, 307). As the infrared spectra of complexes I and II are very similar, only the copper complex spectrum of doxycycline and phenanthroline (Figure 4) will be discussed.

The infrared spectrum of doxycycline shows characteristic absorptions at 3426 and 3200 cm-1, corresponding to vOH and vNH, respectively. These absorptions overlap in the complex, producing a broadband centered at about 3370 cm-1. The amide band, v (C = 0), which appears on the doxycycline spectrum at 1670 cm-1, cannot be distinguished on the complex spectrum, indicating that the amide group oxygen is involved in the coordination sphere. strong, at 1618 and 1578 cm-1, which are attributed to carbonyl stretches of rings A and C, v (C = O), appear at the same wavelength, discarding the participation of these oxygen in the coordination sphere. The poorly coordinated perchlorate ion behavior or as counterion can be distinguished by the frequencies of vCl-O. The lowering of symmetry from Td to C3v, which occurs when one of the perchlorate oxygen binds to another atom, induces the unfolding of v3 in two bands. Three intense bands at 1121, 1108 and 1087 cm-1 appear in the complex spectrum, which can be explained by the presence of one unidentified and one free perchlorate ion.

Spectrum analysis in the IV region for complex III, which can be seen in Figure 5, confirmed the coordination of hydf in bidentate to metal form. The free hydf has an absorption at 1657 cm "1, referring to vC = O which shifts to 1645 cm" 1 in the complex. A change in vNH absorbance from 3257 and 3147 cm -1 in the free binder to 3149 and 3079 cm -1 in the complex can also be seen. The coordination of metal to phen can be confirmed by the presence of the 1599 cm-1 band for the vC = NH stretch.

The positive mode ESI-MS spectra show, for complexes I and II, a major peak in m / z 686, characteristic of an ion containing 63CuZ65Cu (2.2%), attributed to the monocarloaded species [Cu (dox) (phen) -H +] + or [Cu (tc) (phen) -H +] + (calculated mass = 686.14), which can be seen in Figure 6. In the spectra of the two complexes, a lower peak intensity in m / z 785 , 9 corresponds to the species [Cu (dox) (phen) (ClO4)] + or [Cu (tc) (phen) (ClO4)] + (calculated mass = 786.1).

The structure of complex III, determined by single crystal X-ray crystallography, revealed a distorted octahedral geometry around the copper atom, shown in Figure 7. The complex crystallizes in the triclinic space group P-1 with a = 9.8120 (2 ) A, b = 11.2001 (3) A, c = 11.3551 (2) A, α = 104.564 (2) °, β = 100.049 (2) °, γ = 90.218 (2) °. The equatorial positions are occupied by hydf, which is bound to bidentate copper via nitrogen and carbonyl oxygen and to 1,10-phenanthroline which binds to the metal via two heterocyclic nitrogen. The axial sites are occupied by a perchlorate oxygen and acetonitrile nitrogen. The second perchlorate anion acts as a counterion of the complex and is stabilized by hydrogen bonds.

C) Evaluation of antitumor activity of copper complexes

Example 3: K562 cell growth inhibition

Cell line K562 was purchased from the Rio de Janeiro Cell Bank (CR083 RJCB collection number). This cell line was created from the pleural effusion of a 53-year-old woman with chronic myeloid leukemia in terminal blast crisis. Cells were cultured in RPMI 1640 medium (Sigma Chemical Co.) supplemented with 10% fetal bovine serum (CULTILAB, Sao Paulo, Brazil) at 37 ° C in a 5% CO2 atmosphere. Cell viability was verified by Trypan blue. Cell number was determined by Coulter automatic counter analysis. For cytotoxicity evaluation, 1 χ 10 ^ 5 cells mL "1 were incubated for 72 h in the absence and presence of increasing concentrations of the tested complexes. The sensitivity of cells to the complexes was evaluated by concentration that inhibits cell growth by 50%. IC50- The values obtained are presented in Table 3.

Table 3: Inhibition of K562 cell growth by complexes I, II and III.

<table> table see original document page 11 </column> </row> <table>

Complexes I, II and III inhibited K562 cell growth with Cl50 values of 1.93, 2.59 μmol L1 and 2.15 μmol L "1, respectively. Complex I is the most active among them and In all cases, the activities of the complexes were superior to those of free ligands.

Example 4: Intracellular Accumulation of Tested Complexes

K562 cells (1x10 ^ 5 cells mL "1) were incubated for 72 h in the absence and presence of increasing complex concentrations. After incubation, an aliquot was taken, washed three times with ice isotonic buffer solution and the pellet resuspended in a 33% HNO3 solution.Copper concentration was determined by graphite furnace atomic absorption spectrometry.The data presented in Figure 8 allow us to conclude that there is a good correlation between cytotoxic activity and intracellular accumulation, ie inhibition of cell growth increases with increasing intracellular concentration of the complex.

Example 5: Evaluation of interactions of new complexes with DNA

In order to verify that the complexes bind to DNA within cells, they were incubated for 2 hours with increasing concentrations of complexes I and II (Figure 9a), the DNA was extracted according to the protocol described in the literature (NA Rey, A. Neves, PP Silva, FCS Paula, JN Silveira, FV Botelho, LQ Vieira, CT Pich, H. Terenzi, EC Pereira-Maia, J. Inorg. Biochem. 103, 2009, 1323) and the formed adducts were quantified. Nucleotide DNA concentration was determined by spectrophotometry (ε = 6600 molL ^ 1cm ^ 1 at 260 nm). The absorbance ratio at 260 and 280 nm was between 1.6-1.9. Copper was measured by graphite furnace atomic absorption spectrometry. The number of adducts formed grows with increasing complex concentration. A similar study was performed for complex III (Figure 9b) with an incubation time of 72 hours.

The interactions of the complexes with DNA were also studied by UV-Vis spectrometry (Figure 10). Solution spectra of the complexes in the absence and presence of increasing concentrations of calf thymus DNA were recorded. The addition of DNA induces a hypochromic effect, indicating that the tested compounds form ternary complexes with DNA. To compare the stability of the complexes, the affinity constant, K, was calculated from spectrophotometric data according to the equation below:

[DNA] / (Ea - Bf) = [DNA] / (B0 - 8f) + 1 / K (B0 - 8f)

where [DNA] is the concentration of DNA in base pairs, s is the ratio of absorbance / [Pt], sf is the extinction coefficient of the free complex and B0 is the extinction coefficient of the complex in fully bound form. The slope to intersection ratio of the [DNA] / (sa - Bf) versus [DNA] plot gives the value of the complex affinity constant for DNA. The calculated affinity constants were 2.02 χ 10 ^ 4; 2.95 χ 10 ^ 4 and 2.19 χ 10 ^ 4 for complexes I, II and III, respectively. Complex II has slightly higher DNA affinity than the others.

Description of the figures

Figure 1 presents the proposed chemical structures for complexes I, II and III.

Figure 2 shows the electronic paramagnetic resonance spectra of complexes I and III.

Figure 3 shows the UV-Vis absorption spectra of 1.6 χ 10 ^ 4 moIL ^ 1 methanolic solutions of: (a) phen, dox and complex I; (b) phen, tc, complex II and (c) phen, hydf and complex III.

Figure 4 shows the IR region spectra for: (a) dox and complex I and (b) tc and complex II.

Figure 5 shows the spectrum in the IV region for complex III.

Figure 6 shows the electrospray mass spectrum: (a) complex II spectrum dissolved in methanol: water (1: 1) solution and (b) isotopic distribution calculated from the Qual Browser version 2.0.7 copyright® program.

Figure 7 shows the ORTEP representation of complex III.

Figure 8 shows the percentages of cell survival after 72 hour incubation with equotoxic concentrations of compounds I and II as a function of intracellular copper concentration.

Figure 9 shows the amount of Cu-DNA adducts as a function of complex concentration: (a) complexes I and II, incubation for 2 h and (b) complex III, incubation for 72 h.

Figure 10 shows the result of titration of the II complex with DNA. [[Cu (dox) (phen) (H20) (IC04)] (IC04)] = 2 χ 10 "5 molL" 1, [DNA] 0 to 3 χ 10 "4 molL" 1.

Claims (9)

1. Copper complexes, characterized in that they have the following formula <formula> formula see original document page 14 </formula> where R = tetracycline, doxycycline, minocycline, tigecycline, benzoic acid hydrazides, furic acid hydrazides; R 1 = 1,10-phenanthroline, 2,9-dimethyl-1,10-phenanthroline, 4,7-diphenyl-1,10-phenanthroline; and R2 = water, acetonitrile. Copper complexes according to Claim 1, characterized in that it has a concentration necessary to inhibit 50% of cell growth (IC50) in K562 strain, less than 3 pmol L "1. Pharmaceutical composition, characterized in that it comprises a copper complex II according to the following structural formula: where R = tetracycline, doxycycline, minocycline, tigecycline, benzoic acid hydrazides, acid hydrazides furo; R 1 = 1,10-phenanthroline, 2,9-dimethyl-1,10-phenanthroline, 4,7-diphenyl-1,10-phenanthroline; R2 = water, acetonitrile; and at least one pharmaceutically acceptable excipient. 4. A process for the synthesis of copper complexes, comprising: (a) adding a copper (II) solution to a solution of an antibiotic selected from the tetracycline family or to a solution of a furic acid hydrazide or benzoic acid; b) adding a solution of a phenanthroline to the mixture obtained in step a; c) filtration, washing with water and drying of the solid obtained. Copper complex synthesis process according to Claim 4, characterized in that the copper solution is preferably copper (II) perchlorate solution. Copper complex synthesis process according to Claim 4, characterized in that the tetracycline family antibiotic is selected from the group comprising doxycycline, tetracycline, minocycline and tigecycline. Copper complex synthesis process according to Claim 4, characterized in that phenanthroline is selected from the group comprising 1,10-phenanthroline; 2,9-dimethyl-1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline. Process for synthesizing copper complexes with antitumor activity according to claim 4, characterized in that the phenanthroline solubilization comprises an organic solvent, preferably methanol or acetonitrile. 9. Copper complexes, characterized in that it is in the preparation of an antitumor drug.