Imidazolinylhydrazones have high levels of biological activity [1, 2]. A series of hydrazones of 2-hydrazinoimidazoline have antimicrobial, antifungal, cytotoxic, and antitumor activity [3-7]. The present report describes results obtained from studies of the biological activities of a series of new 2-imidazolinylhydrazones of mono- and dicarboxylic compounds of types I – IX.

Compounds I – IX were prepared by interaction of the corresponding aldehydes with 2-hydrazinoimidazoline hydrobromides in ethanol in the presence of an equimolar quantity of KOH [8]. All compounds were stable crystalline solids; their structures were established by IR, 1H NMR, and mass spectroscopy. The mass spectra of hydrazones I-IX contained peaks corresponding to the molecular ions. The presence of singlet signals from the four methylene group protons in the 1H NMR spectra of all compounds suggests that they exist in the diazine form due to transfer of a hydrazine proton to the imidazoline nitrogen atom [79].

Experimental Chemical Section

IR spectra were recorded on a Varian Scimitar 100 FT-R spectrophotometer at 400 – 4000 cm–1; samples were prepared as suspensions in Vaseline grease. PMR spectra were recorded in DMSO-d6 on a Varian Unity 300 (300 MHz) spectrometer. Mass spectra were taken on a Bruker Autoflex II spectrometer with electrospray ionization. Hydrazones were synthesized using commercially available hydrazinoimidazoline hydrobromide (Aldrich). Elemental analysis data for the compounds synthesized corresponded to calculated values.

Hydrazones I – IX were prepared using our previously reported method [8].

2-((Imidazolidin-2-ylidenhydrazono)methyl)phenol (I). The yield was 60%. The melting temperature was >260°C. The atomic formula was C10H12N4O. The IR spectrum (ν, cm-1), was: 3250 (OH), 3172 (NH), 1670, 1601 (C=N). The PMR spectrum in DMSO-d6 (δ, ppm), was: 11.158 (s, 1H, OH); 8.234 (s, 1H, CHazomet); 7.42 (d, 1H, J 7.8 Hz, CHarom); 7.20 (broad s, 2H, NH); 7.13 – 7.18 (m, 1H, CHarom); 6.80 – 6.85 (m, 2H, CHarom); 3.456 (s, 4H, CH2). The mass spectrum, m/z, was: 204, 187, 177.

2-((Imidazolidin-2-ylidenhydrazono)methyl)-4-metho xyphenol (II). The yield was 55%. The melting temperature was 205°C. The atomic formula was C11H14N4O2. The IR spectrum (ν, cm-1), was: 3245 (OH), 3178 (NH), 1660, 1598 (C=N). The PMR spectrum in DMSO-d6 (δ, ppm), was: 10.983 (s, 1H, OH); 8.088 (s, 1H, CHazomet); 6.67 – 6.77 (m, 3H, CHarom); 6.580 (s, 1H, NH); 6.485 (s, 1H, NH); 3.718 (s, 3H, CH3); 3.444 (s, 4H, CH2). The mass spectrum, m/z, was: 234, 217, 203.

4-Bromo-2-((imidazolidin-2-ylidenhydrazono)methyl)-phenol (III). The yield was 70%. The melting temperature was >260°C. The atomic formula was C10H11BrN4O. The IR spectrum (ν, cm-1), was: 3235 (OH), 3180 (NH), 1665, 1600 (C=N). The PMR spectrum in DMSO-d6 (δ, ppm), was: 11.539 (s, 1H, OH); 8.085 (s, 1H, CHazomet; 7.39 (d, 1H, J 2.4 Hz, CHarom); 7.17 (dd, 1H, J1 9.0 Hz, J2 2.4 Hz, CHarom); 6.75 (d, 1H, J 8.7 Hz, CHarom); 6.696 (s, 1H, NH); 6.575 (s, 1H, NH); 3.451 (s, 4H, CH2). The mass spectrum, m/z, was: 283, 266, 203.

4- tert -Butyl-2-((imidazolidin-2-ylidenhydrazono)methyl) phenol (IV). The yield was 80%. The melting temperature was 215°C. The atomic formula was C18H28N4O. The IR spectrum (ν, cm-1), was: 3270 (OH), 3190 (NH), 1672, 1602 (C=N). The PMR spectrum in DMSO-d6 (δ, ppm), was: 12.049 (s, 1H, OH); 8.180 (s, 1H, CHazomet); 7.10 – 7.14 (m, 2H, CHarom); 6.604 (s, 1H, NH); 6.530 (s, 1H, NH); 3.374 (s, 4H, CH2); 1.392 (s, 9H, t-Bu); 1.239 (s, 9H, t-Bu). The mass spectrum, m/z, was: 316, 299, 242.

N-(2-((Imidazolidin-2-ylidenhydrazono)methyl)-phenyl)-4-methylbenzosulfonamide (V). The yield was 45%. The melting temperature was 225°C. The atomic formula was C17H19N5O2S. The IR spectrum (ν, cm-1), was: 3314, 3140 (NH), 1657 (C=N), 1332 (SO2)as, 1154 (SO2)s. The PMR spectrum in DMSO-d6 (δ, ppm), was: 11.55 (broad s, 1N, NH); 8.228 (s, 1H, CHazomet); 7.58 – 7.65 (m, 3H, CHarom); 7.40 (broad s, 2H, NH); 7.32 (d, 2H, J 9.0 Hz, CHarom); 7.02 – 7.18 (m, 3H, CHarom); 3.556 (s, 4H, CH2); 2.494 (s, 3H, CH3). The mass spectrum, m/z, was: 357, 329.

2-((2-(Diphenylphosphino)benzyliden)hydrazono)-imidazolidine (VI). The yield was 60%. The melting temperature was 252°C. The atomic formula was C22H21N4P. The IR spectrum (ν, cm-1), was: 3215 (NH), 1645, 1598 (C=N). The PMR spectrum in DMSO-d6 (δ, ppm), was: 8.180 (d, 1H, JP 5.4 Hz, CHazomet); 8.57 (broad s, 2H, NH); 8.19 – 8.22 (m, 1H, CHarom); 7.42 – 7.52 (m, 8H, CHarom); 7.16 – 7.22 (m, 4H, CHarom); 6.80 – 6.84 (m, 1H, CHarom); 3.686 (s, 4H, CH2). The mass spectrum, m/z, was: 372, 344, 288.

3-(Imidazolidin-2-ylidenhydrazono)-2-methylbutanal oxime (VII). The yield was 65%. The melting temperature was >260°C. The atomic formula was C7H13N5O. The IR spectrum (ν, cm-1), was: 3330 (OH), 3195 (NH), 1654, 1595 (C=N). The PMR spectrum in DMSO-d6 (δ, ppm), was: 10.917 (s, 1H, NOH); 6.553 (s, 2H, NH); 3.341 (s, 4H, CH2); 1.982 (s, 3H, CH3); 1.953 (s, 3H, CH3). The mass spectrum, m/z, was: 183, 152, 137.

4- tert -Butyl-2,6- bis -((imidazolidin-2-ylidenhydrazono)-methyl)phenol (VIII). The yield was 75%. The melting temperature was 257°C. The atomic formula was C18H25N8O. The IR spectrum (ν, cm-1), was: 3308 (OH), 3170 (NH), 1660, 1625 (C=N). The PMR spectrum in DMSO-d6 (δ, ppm), was: 12.188 (s, 1H, OH); 8.364 (s, 2H, CHazomet); 7.622 (s, 2H, CHarom); 7.45 (broad s, 4H, NH); 3.529 (s, 8H, CH2); 1.293 (s, 9H, t-Bu). The mass spectrum, m/z was: 370, 353, 294.

2,6- bis -(1-(Imidazolidin-2-ylidenhydrazono)-ethyl)pyridine (IX). The yield was 70%. The melting temperature was >260°C. The atomic formula was C18H25N8O. The IR spectrum (ν, cm-1), was: 3324 (NH), 1650, 1595 (C=N). The PMR spectrum in DMSO-d6 (δ, ppm), was: 8.60 (broad s, 4H, NH); 8.364 (d, 2H, J 8.1 Hz, CHarom); 7.622 (t, 1H, J 8.1 Hz, CHarom); 3.764 (s, 6H, CH3); 2.434 (s, 8H, CH2). The mass spectrum, m/z, was: 327, 312.

Experimental Biological Section

Protistocidal activity was studied as described in [10] using the protozoa Colpoda steinii (a field isolate, collection of the Parasitology Laboratory, North Caucasus Zonal Science Research Veterinary Institute). The maximum test concentration was 500 μg/ml.

Antibacterial activity was evaluated using Staphylococcus aureus P-209 (a field isolate obtained in April 2012, museum of the Russian Regional Veterinary Laboratory) and Escherichia coli O39 (a field isolate obtained in August 2008, museum of the Rostov Regional Veterinary Laboratory) by serial twofold dilutions on liquid nutritive medium with a microbial load of 2.5 × 105 cells/ml [11]. For preparation of bacterial cultures, one-day agar cultures were transferred with a platinum loop to a tube containing physiological saline (3 ml) and an initial dilution to 500 × 106 microbial cells/ml was prepared using an optical standard. The resulting suspension was diluted with Hottinger broth initially 100-fold and then a further 10-fold to obtain a suspension containing 500,000 microbial cells/ml, and this was used as the working dilution of the culture. The maximum test concentration was 250 μg/ml. Aliquots (1 ml) of the prepared cultured were transferred to tubes containing dilutions of compounds, as well as to controls not containing substances. The reference substances were Baycox (solution; from Bayer HealthCare AG, Germany) for assessment of protistocidal activity and furazolidone (tablets; from OAO Borisovskii Medicines Factory, Belarus) for assessment of antibacterial activity. Initial dilutions of test compounds were prepared by mixing 5 mg of substance, 50 μl of DMSO (90%), and 5 ml of distilled water to make the first dilution with a concentration of 1000 μg/ml. Further dilutions were prepared in liquid nutritive medium. Concentrations are given in Table 1.

Table 1 Protistocidal and Antibacterial Activity of Hydrazones I – IX

The active dose was taken as the minimum inhibitory concentration (MIC) leading to suppression of test microorganism growth.

Compounds IV, V, and IX were found to have high protistocidal activity, 10 – 15 times greater than that of Baycox, which is used for the treatment of coccidiosis in poultry [10], while 2-hydrazinoimidazoline itself lacked protistocidal activity (MIC > 500 μg/ml). These results support the value of seeking protistocidal agents among the imidazolinylhydrazones. The study compounds did not have antibacterial activity. At this stage of the study, we can conclude that the 2-imidazolinylhydrazone fragment itself does not have high protistocidal activity. The activity of compounds IV and V may arise as a result of their surfactant properties, due to the fact that these molecules contain hydrophobic aldehyde fragments and hydrophilic 2-imidazoline heterocycles. There is also potential in continuing the search among pyridine derivatives related to compound IX.