BRPI1002523B1 - SYNTHESIS PROCESS OF COPPER COMPLEXES WITH ANTITUMORAL ACTIVITY - Google Patents

Abstract

synthesis of copper complexes with antitumor 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 by the furic acid or the 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

“SYNTHESIS PROCESS OF COPPER COMPLEXES WITH ANTITUMORAL ACTIVITY”

Field of invention

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 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.

State of the art

The search for metal complexes with antitumor properties began with the discovery of the antitumor action of cisplatin, cisdiaminodichloroplatin (ll), by Rosenberg and Van Camp in the late sixties (B. Rosenberg, L. Van Camp, JE Trosco, VH Mansour , Nature, 222,1969, 385) and has been stimulated with the discovery of the 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 great impact, enabling the complete cure of several tumors, particularly the testicular and ovarian (B. Rosenberg, L. Van Camp, Cancer Research, 30, 1970, 1799). Unfortunately, despite the therapeutic success of cisplatin, it has some disadvantages such as low water solubility, undesirable side effects, in addition to the appearance of cell resistance. Therefore, there is a search for compounds that present better solubility, have less side effects and activity in cisplatin-resistant tumors.

DNA is the main target of antitumor drugs. Metal complexes can bind directly to DNA, as is the case with cisplatin (A.M.J.

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Fichtinger-Schepman, J.L. Van der Veer, J.H.J. den Hartog, P.H.M. Lohman, J. Reedijk, Biochemistry, 24, 1985, 707), or indirectly, through oxy-reduction reactions, generating active species that can break the DNA molecule, such as bleomycin, a natural and potent 5 glycopeptide. antitumor (J. Reedijk, Proc. Natl. Acad. Sci. USA, 100, 2003, 3611).

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

On the other hand, the compound 1,10-phenanthroline and its derivatives are of interest in the synthesis of new metallopharmaceuticals with antitumor activity, since they have flat structures and, therefore, are capable of interleaving in the DNA strand and preventing the cell replication. Another advantage of these compounds is the absence of antibacterial activity against the intestinal flora, which reduces the side effects of a possible metallopharmaceutical with these 15 compounds (S. Roy, K.D. Hagen, P.U. Maheswari, M. Lutz, A.L. Spek, J.

Reedijk, G.P. van Wezel, Chem. Med. Chem., 3, 9, 2008, 1427).

A well-studied example is the Cu (ll) complex of 1.10phenanthroline. The reactive species [Cu (Phen) 2] ⁺ made an oxidative attack on a ribose carbon. Hydrogen peroxide can be generated by reducing oxygen by the species [Cu (Phen) 2] ⁺ (T. Hirohama, Y. Kuranuki, E. Ebina, T.

Sugizaki, H. Arii, M. Chikira, P.T. Selvi, M. Palaniandavar, J. Inorg. Biochem., 99, 2005, 1205).

Some patents containing metallic compounds with 1,10-phenanthroline derivatives that have anti-tumor activities are available in the literature. As examples, two American patents can be cited: 4,699,978 (Site-specific chiral ruthenium (II) and cobalt (III) antitumor agents) and 5,225,556 (Chemical probes for left-handed DNA and for A-DNA; chiral metal complexes as Z-specific antitumor agents and double strand cleavers) that comprise syntheses, characterizations and 30 cytotoxic studies of complexes with 1,10-phenanthroline derivatives. They were shown to be effective in breaking the DNA molecule and consequently preventing the proliferation of tumor cells. They can also

3/12 two Chinese patent applications registered with the European patent office: CN101190925 (Copper complex, preparation method and application) and CN101225092 (Manganese complex as well as preparation method and uses) which describe the syntheses, characterizations and applications 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 a broad spectrum of action, low toxicity, oral administration and low cost. However, this family of compounds has other pharmacological properties in addition to antimicrobial activity. Some patents containing tetracyclines and analogs as compounds capable of inhibiting the growth of cancer are already available in the literature. As examples, two American patent application documents can be cited: WO / 1998/031224 (Method of inhibiting cancer growth) which comprises the use of a tetracycline analogue to induce cytotoxic effects against cancer in mammals, being particularly effective in inhibiting the 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 the treatment of tumors and neoplastic diseases with the combination of antitumor drugs and tetracycline, so that it inhibits protein synthesis in the target cell.

The perspective of the pharmaceutical use of tetracycline or derivatives complexed with metals, too, is another aspect of studies. American patent application WO / 2003/066064 (Methods of disease treatment using metal-complexed tetracycline antibiotics) describes the application of tetracycline compounds and quinones complexed 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,

4/12 who are involved in different stages of cancer progression. It has been suggested that this inhibition is related to the coordination of protein zinc or calcium atoms (RA Garcia, DP Pantazatos, CR Gessner, KV Go, VL Woods Jr., FJ Villarreal, Mol. Pharmacol. 67, 4, 2005,1128 ).

In addition, recent studies show that platinum (II) complexes with doxycycline and tetracycline inhibit the growth of chronic myeloid leukemia cells and this antitumor activity is possibly related to the complex's interaction with DNA by intercalation 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 anti-tumor properties of the compound [Cu (Phen) ₂ ] ^{2+ with} those of hydrazide derivatives, since the latter also have neoplastic properties. Regarding the antitumor activity of hydrazide derivatives, we can mention the Brazilian patent application PI0512526-0 (Compound, pharmaceutical composition, method to treat a cancer patient and method to prepare a bis (thiohydrazide amide) di-salt) that suggests the use of a pharmaceutical composition comprising a salt derived from hydrazide, salt of bis (thiohydrazide amide), 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, is less toxic than platinum. The binders employed can be tetracycline (tc), doxycycline (dox), minocycline (min), tigecycline (tig), all belonging to the tetracycline family, hydrazides of benzoic acid (hydb) or furic acid (hydf ), as well as 1,10-phenanthroline (phen) or its derivatives, 2,9-dimethyl-1,10-phenanthroline (neocuprine) (2,9-dmphen) or 4,7-diphenyl-1,10phenanthroline (batophenanthroline) (4,7-dfphen).

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The present invention proposes the synthesis of novel compounds that can be used in pharmaceutical formulations to treat cases of neoplasms. The structures of three of the compounds [Cu (R) (Ri) (R2) (CIO4)] (CIO4), where R = tc, dox, min, tig, hydf, hydb; Ri = phen, 2,9-dmphen, 4,7-dfphen and R ₂ = H ₂ O, acetonitrile, are shown in Figure 1. The compounds were synthesized and characterized through elemental and conductometric analysis, electronic paramagnetic resonance (EPR) , ultraviolet-visible (UVVis) and infrared (IV) spectrometry, mass spectrometry and monocrystal X-ray crystallography, for complex III.

All compounds have significant cytotoxic activity in tumor cell lines and may be used in the treatment of cancer.

The compositions of the present invention are characterized by the use of copper complexes combined with pharmaceutically acceptable excipients. Standard compositions can be liquid and solid. Since liquid preparations can be presented as a solution, suspension or emulsion. Solids in the form of capsules, tablets, pills or tablets.

These compositions can be administered intramuscularly, intravenously, orally or as devices that can be implanted or injected.

The technology can be better understood by analyzing the following, non-limiting examples:

A) Synthesis of complexes

Example 1 - Synthesis of complexes I and II:

Complex I was prepared by reacting Cu (CIO4) ₂ -6H ₂ O (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 methanol solution of 1.10-phenanthroline (0.0996g, 0.5 mmol) was added. The resulting mixture was stirred for another three hours. The formed precipitate was filtered off, washed with water and dried in vacuo. A green solid of composition [Cu (C ₂₂ H ₂ 4N ₂ O8) (Ci ₂ H8N ₂ ) (H ₂ O) (CIO4)] (CIO4) was obtained.

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Complex II was prepared in a similar way, with the addition of tetracycline instead of doxycycline.

Example 2 - Synthesis of complex III:

Complex III was prepared by reacting Ου (ΟΙ0 ₄ ) 2 · 6Η2θ (0.1853g, 0.5 mmol) with 2-furic acid hydrazide (0.2405g, 0.5 mmol), in acetonitrile (5 mL), followed by the addition of a solution of 1.10-phenanthroline (0.0996g, 0.5 mmol) in acetonitrile (3 mL). Blue crystals of composition [Cu (C5H ₆ N2O2) (C ₁ 2H8N2) (C2H ₃ N) (CIO4)] (CIO4) were obtained.

It is also possible to obtain a similar complex, using water as a solvent instead of acetonitrile, whose final composition is [CU (C5H6N2O2) (C ₁₂ H ₈ N2) (H2O) (CIO4)] (CIO4).

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:

Cu ²⁺ (sol1) ⁺ L ¹ (sol1) -> CuL ¹ + L ² (soI2) —► C |, C || or C |||

The complex I (C |) = [CuL ¹ L ² (CIO4) (H2O)] CIO4, when sol1 = water and sol2 = methanol, L ¹ = dox and L ² = phen; complex II (Cn) = [CuL ¹ L ² (CIO4) (H2O)] CIO4, when sol1 = water and sol2 = methanol, L ¹ = tc and L ² = phen and complex III (Cm) = [CuL ¹ L ² (CIO4) (CH3CN)] CIO4, when sol1 = water and sol2 = acetonitrile, L ¹ = hydf and L ² = phen.

The compounds were characterized by elemental analysis, conductivity, EPR, IV and UV-Vis spectrometry, mass spectrometry (complexes I and II) and monocrystalline X-ray crystallography (complex III). The data of elementary analysis and molar conductivity are reported in Table 1 and the data of RPE are reported in Table 2.

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Table 1: Data from elementary and conductometric analyzes for complexes I, II and III. Values in parentheses are calculated for [Cu (Ci2H8N2) (C22H24N2O8) (H ₂ O) (CIO4)] (CIO4), complexes I and II and [Cu (C ₅ H ₆ N2O2) (Ci2H8N2) (C2H ₃ N) (CIO4)] (CIO4), complex III.

Complexes %Ç %H % N %Ass Λμ (gS / cm) Complex I MM = 905.11 g.mol · ¹ 45.00 (45.12) 3.82 (3.79) 6.11 (6.19) 6.98 (7.02) 173.9 Complex II MM = 905.11 g.mor ¹ 45.63 (45.12) 4.01 (3.79) 6.49 (6.19) 7.12 (7.02) 187.9 Complex MM MM = 609.55 g.mol · ¹ 37.87 (37.42) 2.49 (2.81) 11.14 (11.49) 10.36 (10.83) 202.9

The elementary 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: EPR parameters for complexes I, II and III.

Compounds EPR parameters g // A // (G) A // (10 · * cm) Qnl IA // I (cm) Complex 1 2.63 2,271 172.3 182.7 124.3 Complex II 2,063 2,271 172.3 182.7 124.3 Complex III 2,062 2.256 173.6 182.8 123.4

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

The analysis of the absorption spectra in the UV-Vis region, for complexes I and II, suggested the involvement of ring A of tc and dox in metal coordination. The absorption band at 269 moved to 293 nm in complex I, as shown in Figure 3a, and from 270 to 296 nm in

8/12 complex II (Figure 3b), while the band centered at 362 nm for the chromophore BCD of the tc or dox did not change in both complexes. For complex III, the phen band centered at 264 nm undergoes a bathochromic shift to 272 nm in the complex indicating the complexation of the ligand to the metal (Figure 3c).

The assignments of absorptions in the infrared spectra of complexes I and II were made based on a detailed study of tetracyclines and analogues by Dziegielewski et al. (J. Dziegielewski, J. Hanuza, B. Jezowska-Trzebiatowska, Bull Acad. Pol. Sci., Ser. Sci. Chim. 24, 1976, 307). As the infrared spectra of complexes I and II are very similar, only the spectrum of the doxycycline and phenanthroline copper complex (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 are superimposed on the complex, producing a broadband centered around 3370 cm ' ¹ . The amide band, v (C = O), which appears in the doxycycline spectrum at 1670 cm ' ¹ , cannot be distinguished in the complex spectrum, indicating that oxygen from the amide group is involved in the coordination sphere. Two very strong bands, in 1618 and 1578 cm ' ¹ , which are attributed to stretches of the carbonyls of rings A and C, v (C = O), appear in the same wave number, discarding the participation of these oxygenates in the coordination sphere. The behavior of the weakly coordinated perchlorate ion or as a counterion can be distinguished by the vCI-O frequencies. The lowering of symmetry from Td to C3 _V , which occurs when one of the perchlorate oxygen attaches to another atom, induces the V3 split in two bands. Three intense bands in 1121, 1108 and 1087 cm ' ¹ appear in the complex's spectrum, which can be explained by the presence of a unidentated perchlorate ion and a free one.

The analysis of the spectrum in the IV region for complex III, which can be seen in Figure 5, confirmed the coordination of the hydf in a bidentified way to the metal. The free hydf has an absorption of 1657 cm ' ¹ , referring to vC = O which moves to 1645 cm' ¹ in the complex. It can also be verified a

9/12 change in absorbances referring to 3257 and 3147 cm ' ¹ vNH in the free ligand to 3149 and 3079 cm' ¹ in the complex. The coordination of the metal to the phen can be confirmed by the presence of the band at 1599 cm ' ¹ referring to the stretch vC = NH.

The ESI-MS spectra in positive mode show, for complexes I and II, a main peak in m / z 686, characteristic of an ion containing ⁶³ Cu / ® ⁵ Cu (2.2%), attributed to the mono-charged 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 peak of lower intensity in m / z 785.9, corresponds to the species [Cu (dox) (phen) (CIO4)] ⁺ or [Cu (tc) (phen) (CIO4)] ⁺ (calculated mass = 786.1).

The structure of complex III, determined by monocrystalline 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 linked to copper in a bidentate form via nitrogen and carbonyl oxygen and to 1.10-phenanthroline which binds to metal via two heterocyclic nitrogens. The axial sites are occupied by a perchlorate oxygen and acetonitrile nitrogen. The second perchlorate anion acts as a counter ion of the complex and it is stabilized by hydrogen bonds.

C) Evaluation of the antitumor activity of copper complexes

Example 3: K562 cell growth inhibition

The K562 cell line was purchased from the Rio de Janeiro Cell Bank (CR083 number of the RJCB collection). This cell line was created from a pleural effusion of a 53-year-old woman with chronic myeloid leukemia in terminal blast crisis. The cells were cultured in RPMI 1640 medium (Sigma Chemical Co.) supplemented with 10% fetal bovine serum (CULTILAB, São Paulo, Brazil), at 37 ° C, in an atmosphere at 5% CO ₂ . Cell viability was verified by Trypan blue. The number of cells was determined by analysis using a Coulter automatic counter.

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For cytotoxicity evaluation, 1 x 10 ⁵ mL ' ¹ cells were incubated for 72 h in the absence and in the presence of increasing concentrations of the tested complexes. The sensitivity of cells to complexes was evaluated by the concentration that inhibits cell growth by 50%, IC50. The 5 values obtained are shown in Table 3.

Table 3: Inhibition of K562 cell growth by complexes I,

II and III.

Compound IC50 (pmol L ' ¹ ± sd) dox 17.70 ± 0.9 tc 52.37 ± 3.1 hyd 455.62 ± 20.9 Phen 3.17 ± 0.25 [Cu (Phen) ₂ ] (CIO ₄ ) 2 3.44 ± 0.30 Complex I 1.93 ± 0.18 Complex II 2.59 ± 0.26 Complex III 2.15 ± 0.23

Complexes I, II and III inhibited the growth of K562 cells with the 10 IC ₅₀ values of 1.93, 2.59 μπιοΙ L ' ¹ and 2.15 μηηοΙ L' ¹ , respectively. Complex I is the most active among them and, in all cases, the activities of the complexes were superior to that of free ligands.

Example 4: Intracellular accumulation of the complexes tested

K562 cells (1x10 ⁵ cells mL ' ¹ ) were incubated for 72 h in the absence and in the presence of increasing concentrations of the complexes. After incubation, an aliquot was taken, washed three times with cold isotonic buffer solution and the pellet resuspended in a 33% HNO _{3 solution} . The copper concentration was determined by atomic absorption spectrometry in a graphite furnace. The data presented in Figure 8 allow

11/12 conclude that there is a good correlation between cytotoxic activity and intracellular accumulation, that is, the inhibition of cell growth increases with the increase in the intracellular concentration of the complex.

Example 5: Evaluation of interactions of new complexes with DNA

In order to verify whether the complexes bind to DNA inside the 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. The DNA concentration per nucleotide was determined by spectrophotometry (ε = 6600 moll / cm ¹ at 260 nm). The absorbance ratio at 260 and 280 nm was between 1.6-1.9. Copper was dosed by atomic absorption spectrometry in a graphite furnace. The number of formed adducts grows with the increase in the concentration of complex. A similar study was carried out for complex III (Figure 9b) with an incubation time of 72 hours.

The interactions of complexes with DNA were also studied by UV-Vis spectrometry (Figure 10). Spectra of solutions of the complexes in the absence and in the 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 the DNA. To compare the stability of the complexes, the affinity constant, K, was calculated from the spectrophotometric data, according to the equation below:

[DNA] / (Sa - Cf) = [DNA] / (£ ₀ - 8 _f ) + 1 / K (C ₀ - 8f) where [DNA] is the concentration of DNA in base pairs, s _a is the ratio between absorbance / [Pt], Ef is the extinction coefficient of the free complex and £ θ the extinction coefficient of the complex in the fully bound form. The ratio between the slope and intersection of the graph [DNA] / (s _a - £ f) versus [DNA] gives the value of the DNA affinity constant of the complex. The calculated affinity constants were 2.02 x 10 ⁴ ; 2.95 x 10 ⁴ and 2.19 x 10 ⁴ for

12/12 complexes I, II and III, respectively. Complex II has a slightly higher affinity for DNA than the others.

Description of the figures

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

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

Figure 3 shows the absorption spectra in the UV-Vis region of 1.6 x 10 ' ⁴ molL' 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 spectra in the IV region 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) spectrum of complex II dissolved in methanokagua solution (1: 1) and (b) isotopic distribution calculated from the program Qual Browser version 2.0.7copyright®.

Figure 7 shows the ORTEP representation of complex III.

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

Figure 9 shows the quantity of Cu-DNA adducts as a function of the concentration of complex: (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 complex II with DNA. [[Cu (dox) (phen) (H ₂ O) (CIO ₄ )] (CIO ₄ )] = 2 x 10 ' ⁵ molL' ¹ , [DNA] 0 to 3 x 10 ' ⁴ molL · ¹ .

Claims (6)

1- Copper complexes, characterized by the following structural formula:

where R = tetracycline, doxycycline, tigecycline, minocycline or furic acid hydrazide; R1 = 1.10-phenanthroline; 2,9-dimethyl-1,10-phenanthroline; or 4,7-diphenyl-1,10-phenanthroline; and R2 = water or acetonitrile. 2- Pharmaceutical composition, characterized by comprising the copper complex defined by claim 1 and at least one pharmaceutically acceptable excipient. 3- Process of synthesis of copper complexes defined in claim 1, characterized by comprising the steps: a) adding a copper (II) solution, preferably Cu (ClO ₄ ) ₂ -6H ₂ O, to a solution comprising tetracycline, doxycycline, tigecycline, minocycline or furic acid hydrazide; b) adding a solution of phenanthroline to the mixture obtained in step a; c) filtration, washing with water and drying the obtained solid. 4- Process for the synthesis of copper complexes, according to claim 3, characterized in that phenanthroline is selected from the group comprising 1.10-phenanthroline; 2,9-dimethyl-1,10-phenanthroline and 4,7diphenyl-1,10-phenanthroline. Petition 870190135502, of 12/18/2019, p. 8/9 1/2 5- Process for the synthesis of copper complexes, according to claim 3, characterized by the solubilization of phenanthroline comprising an organic solvent, preferably methanol or acetonitrile. 6- Use of the copper complexes defined in claim 1, characterized by being in the preparation of an anti-tumor medication.