Acquired resistance to antibiotics is currently steadily increasing in microorganisms. Therefore, much attention is being paid to the targeted synthesis of novel biologically active compounds. One group of such drugs comprises thiosemicarbazone derivatives and their metal coordination compounds, many of which exhibit a broad spectrum of biological activity. Antitumor [1], antiviral [2], antibacterial [3], antimalarial [4], and other types of biological activity are known for thiosemicarbazones. Complexation in many instances enhances their biological activity [5,6]. Among the studied compounds, thiosemicarbazones of heterocyclic carbonyl pyridine derivatives occupy a special place [7 – 12]. In particular, the antibacterial and antifungal properties of 2-pyridinealdehyde thiosemicarbazone and certain of its complexes were studied [13 – 18]. Herein experimental results on the synthesis and the study of the bacteriostatic and bactericidal activities of certain derivatives of 8-quinolinealdehyde thiosemicarbazone (HL1-5 and HL6) in addition to their copper coordination compounds of formula Cu(HL1)X2·nH2O, where X = NO3, n = 2 (I); Cl, n = 1 (II); ClO4, n = 3 (III); Cu(L1)NO3 (IV); Cu(L2)NO3·0.25H2O (V); Cu(L3)NO3·H2O (VI); Cu(HL4)(NO3)2·2H2O (VII), Cu(HL5)(NO3)2·2H2O (VIII), and Cu(HL6)(NO3)2·2H2O (IX) are presented in order to find new compounds with antibacterial activity. 8-Quinolinealdehyde is formally structurally similar to heterocyclic 2-pyridinealdehyde. However, it has several important differences. Copper complexes of HL1 were first reported by Ablov, et al. [19]. Considering the processes occurring during purification of the final products obtained by the literature method [19], we improved the synthetic method for the copper coordination complexes with HL1 [20]. The effects of various factors on the biological activity of these compounds were studied. These included coordination and deportonation of the ligand and the nature of the chalcogenide (Y), the substituent on the terminal N atom (R), and the anion (X).

figure a

Composition and structure of copper coordination complexes with HL. Reaction of HL1 with copper nitrate in acidic medium formed Cu(HL1)(NO3)2·2H2O (I). Mixing solutions of Cu(II) chloride or perchlorate and HL1 in the appropriate acid (HCl or HClO4) isolated Cu(HL1)Cl2·H2O (II) and Cu(HL1)(ClO4)2·3H2O (III). Compounds with similar ratios of components were formed by reaction of 2-pyridinealdehyde thiosemicarbazone with the corresponding Cu(II) salts [21,22]. Slow crystallization of an aqueous solution of I formed Cu(L1)NO3 (IV), which was the hydrolysis product of I according to the scheme

Cu(HL1)(NO3)2 + H2O↔Cu(L1)NO3 + H3O+ + NO -3 .

The enhanced protolytic properties of HL1 were an effect of coordination. An x-ray structure of IV [20] showed that the organic ligand was planar and bonded to the central atom, as expected, through N, N, and S donor atoms. The resulting Cu–S coordination bond strongly polarized the NH bond of the five-membered metallocycle and created conditions that were favorable for loss of the proton. The shift of the equilibrium to the right side was also responsible for the lower solubility of IV as compared with I. When an electron-accepting substituent (C6H –5 or p-FC6H –4 ) was added to the terminal N atom, the acid–base properties were so strongly enhanced that complexes with the coordinated neutral thiosemicarbazone became impossible to prepare. Complexes Cu(L2)NO3 (V) and Cu(L3)NO3 (VI) in which the ligand is the anionic form of the corresponding thiosemicarbazone were isolated even from acidic solution. Changing the thiosemicarbazide S atom to O or Se formed Cu(HL4)(NO3)2·2H2O (VII) and Cu(HL5)(NO3)2·2H2O (VIII), where the organic ligand was also coordinated as the neutral species. The ligands in I – VIII were tridentate and coordinated to the Cu ion regardless of the state of deprotonation through N, N, and Y donors (Y = O, S, Se) [20,23]. The ligand with a methylated S (HL6) in complex IX was also tridentate but coordinated through 3 N atoms, forming one six-membered and one five-membered metallocycle. Regardless of the starting metal–ligand ratio and the order of mixing the solutions, products with a Cu–ligand ratio of 1:1 were always obtained. The effective magnetic moments at room temperature for the previously synthesized [19,20,23,24] and newly synthesized complexes (Table 1) fell in the range 1.79 – 1.85 μB and indicated that the Cu had oxidation state +2 and that there was no exchange interaction between the paramagnetic centers. EPR spectra of I – III showed biaxial anisotropy for the g-factors with g || = 2.206, 2.194, and 2.209 and g ⊥ = 2.059, 2.052, and 2.057, respectively. Hyperfine structure was not resolved in the parallel g-factor. The spectrum of VI was isotropic and had a broad line with g iso = 2.114. Because g || > g ⊥> 2.0032, it was assumed that the ground state was the \( {{\text{d}}_{{\text{x}}2 - {\text{y}}2}} \) orbital.

Table 1 Elemental Analyses, % (Found/Calc.) and Effective Magnetic Moments (μef) for Newly Synthesized II, III, VI, and VIII
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Experimental Chemical Part

All HL were synthesized by the literature method [23] in order to prepare 8-quinolinealdehyde semicarbazone starting with 8-quinolinealdehyde and the corresponding chalcogensemicarbazide.

The purity of the products was checked by chromatography on Silufol plates, PMR spectra, and elemental analyses for C, H, and N.

Compounds I, IV, V, VIII, and IX were prepared by the literature methods [20,23,24].

(8-Quinolinidenethiosemicarbazide)copper(II) chloride, Cu(HL 1 )Cl 2 ·H 2 O (II). A solution of CuCl2·2H2O (0.34 g, 2 mmol) in HCl solution (10 mL, 0.1 M) was treated with a solution of HL1 (0.46 g, 2 mmol) in the same acid (15 mL). The dark-green crystals that formed after a few minutes were filtered off and washed with H2O acidifiedwith HCl, EtOH, and Et2O. The compound was very soluble in water and DMF, less soluble in EtOH, and insoluble in Et2O. Yield 0.7 g.

(8-Quinolinidenethiosemicarbazide)copper(II) perchlorate, Cu(HL 1 )(ClO 4 ) 2 ·3H 2 O (III). A solution of Cu(ClO4)2·6H2O (0.37 g, 1 mmol) in HClO4 solution (7 mL, 0.1 M) was treated with HL1 (0.23 g, 1 mmol) in HClO4 (8 mL, 0.1 M). The dark-green crystals that formed were filtered off and washed with H2O acidified with HClO4, EtOH, and Et2O. The compound was very soluble in water, EtOH, and DMF and insoluble in Et2O. Yield 0.36 g.

[8-Quinolinidene-4-(3´-fluorophenyl)thiosemicarbazidato]copper(II) nitrate, Cu(L 3 )NO 3 ·H 2 O (VI). The compound was prepared by mixing a warm (~50°C) solution containing Cu(NO3)2·3H2O (0.24 g) in EtOH (10 mL) and HL3 (0.31 g, 1 mmol) in HNO3 (10 mL) and EtOH (30 mL) at the same temperature. The green crystalline needle-like precipitate that formed on cooling was filtered off, washed with EtOH and Et2O, and dried at room temperature. The compound was slightly soluble in water and EtOH, very soluble in DMF, and insoluble in Et2O. Yield 0.43 g.

(8-Quinolinideneselenosemicarbazide)copper(II) nitrate, Cu(HL 5 )(NO 3 ) 2 ·2H 2 O (VIII). The compound was prepared by mixing solutions of HL5 (0.28 g, 1 mmol) in HNO3 (5 mL, 0.1 M) and Cu(NO3)2·3H2O (0.24 g, 1 mmol) in the same volume of HNO3 (0.1 M). The crystalline precipitate that formed after 4 h was filtered off and washed with H2O acidified with HNO3, EtOH, and Et2O. The compound was very soluble in water and DMF, less soluble in EtOH, and insoluble in Et2O. Yield 0.42 g.

Table 1 presents the elemental analyses and effective magnetic moments for the newly synthesized complexes.

Experimental Biological Part

Antibacterial activity was studied using double serial dilutions [25] in liquid growth medium in meat-peptone bullion. The studied compounds were dissolved in DMF (10 mg/mL). Successive dilutions to the desired concentration were made using meat-peptone bullion.

The test microbes for the in vitro experiment were Gram-positive and Gram-negative microorganisms. The

Gram-positive microbes included five strains of Staphylococcus aureus, three of which were standard (Wood, Smith, 209-P) and two of which were from patients (gem + and a strain from a patient) and standard strains S. saprophyticus, Streptococcus (gr. A, B, G) and Entrecoccus faecalis (strain from a patient). The Gram-negative microbes were standard strains Escherichia coli (str. 0 – 111), Klebsiella pneumoniae, Salmonella typhimurium, S. enteritidis, Proteus vulgaris, P. mirabilis, and Pseudomonas aeruginosa.

Tubes with given concentrations of compounds in growth medium were inoculated with test cultures using 18-hour agar cultures. The seeding dose was 500,000 microbes per mL of medium. The microbe mixture was prepared using the optical turbidity standard.

The activities of the compounds were determined as the minimum concentration inhibiting growth and proliferation of the microorganisms upon exposure for 1 d in a thermostat at 37°C.

Antifungal properties were studied using double serial dilutions in Sabureau bullion using standard strains Aspergillus niger, A. fumigatus, Penicillium, and Candida albicans (strain from a patient). Innoculates were prepared from 7-day fungus cultures. The density of fungus suspensions was determined using bacterial turbidity standard taking into account that the number of fungi was approximately ten times greater than the number of bacteria.

The studied compounds were dissolved in DMF (10 mg/mL). Successive dilutions to the desired concentration were made in Sabureau bullion. Fungistatic activity was determined visually from the lack of fungus growth in growth medium for 14 d at 28°C. Fungicidal activity was determined from the lack of growth after repeated seeding of fungus on Sabureau agar and subsequent incubation for 7 d at 28°C.

Table 2 shows that 8-quinolinealdehyde thiosemicarbazone (HL1) had practically no antibacterial properties. Methylation of the thiosemicarbazide on the S atom (HL6) or replacing the S by O (HL4) did not improve their biological activity against the studied microorganisms. Only for Y = Se (HL5) did substantial activity against Gram-positive bacteria appear.

Table 2 Antimicrobial Activity of Studied Compounds (MIC*/MBC**) (μg/mL)
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Coordination of the thiosemicarbazones HL changed the activity more substantially. Table 2 shows that coordinating HL1 to Cu (I – III) caused a substantial increase of the antibacterial activity against Gram-positive bacteria. These compounds differed in the nature of the anion (NO –3 , Cl–, ClO –4 ). However, a comparison of their antibacterial activities could not confirm that the biological activities of this group of compounds depended on the nature of the anion. The activity of IV, which differed from I by the degree of ligand protonation, was at the same level. Considering the aforementioned protolytic equilibrium and the similarity of the antibacterial activities of I and IV, it could be assumed that the active species was the complex with the anionic form of the ligand. Complexes V and VI also contained a deprotonated ligand but were characterized by greater bacteriostatic activity than that of the unsubstituted analog IV. This indicated that an electron-accepting substituent on the terminal N atom had a favorable influence on the biological activity. This phenomenon was reported previously for the related 2-pyridinealdehyde thiosemicarbazone [14]. The organic ligand in I – VI was bound to the central Cu ion by an N,N,S donor set. It seemed interesting to determine how changing the bonding mode of the organic ligand affected the biological activity of the corresponding copper complexes. Aconvenient subject for this was coordination compound IX, in which the Cu:ligand:nitrate ratio remained at 1:1:2, like in I. The only difference was that the ligand was coordinated through an N,N,N donor set [24]. Table 2 shows that the bacteriostatic activity of IX (against S. aureus str. 209-P and E. faecalis) was lower than that of I. It should be noted that IX typically had slightly greater bacteriostatic and bactericidal activity against Gram-negative bacteria although its activity against Gram-positive bacteria was lower.

Although the biological activity of HL1 changed sharply after complexation to Cu(II), the corresponding semicarbazone HL4 (VII) remained inactive.

The bactericidal activity against S. aureus (str. 209-P) practically did not change upon complexation of selenosemicarbazone HL5 to Cu(II). However, complexation did inhibit somewhat the activity of VIII against E. faecalis (Table 2).

The study of the antifungal activity of chalcogensemicarbazones HL1−6 and their copper complexes I – X showed that they were slightly active against the fungi Candida albicans, A. niger, A. fumigatus, and Penicillium (minimum fungicidal concentration ≥ 600 μg/mL).