The invention relates to new chemical compounds - chiral triazole derivatives, a method of preparation thereof, a pharmaceutical agent containing the new compounds and use thereof. The new compounds exhibit bactericidal and fungicidal activity, finding potential use as effective broad-spectrum antibiotics.

Triazoles represent heterocyclic five-membered aromatic compounds containing three nitrogen atoms. Their beneficial properties have found numerous medical applications. Triazole derivatives are used as bacterial enzyme inhibitors that support antibiotics, e.g. a β-lactamase inhibitor, medicaments inhibiting development of estrogen-dependent cancers, ergosterol synthesis inhibiting medicaments, medicaments inhibiting RNA and DNA replication of some viruses, psychotropic medicaments (such as benzo-l,4-diazepine triazole derivatives) and many others.

Bactericidal activity of triazole derivatives is known from published patent applications US 2005/0113347 or US 2006/0270628. Patents US 4,251,512, 4,147,791, 4,038,406 and 4,048,318 disclose fungicidal compounds and compositions with synergistic activity containing triazole derivatives. Similarly, published international application WO 2006/109933 shows a use of triazole based compounds, their salts and pharmaceutical compositions with fungicidal activity in therapy. Publication WO03/065807 discusses a use of alkoxylated amines for activity enhancement of fungicidal preparations containing fungicidal triazoles.

Patent PL214249 disclosed medically beneficial, antibacterial and antifungal activity of hemiaminals obtained on the basis of 4-amino-4H-l,2,4-triazole. Studies have shown differences in antibacterial and antifungal activity depending on the type of substituents in the phenyl ring. In particular, compounds showing bactericidal activity against Escherichia Coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus Aureus and Candida albicans strains have been disclosed. Nevertheless, the 2bald, 3nbald, 4nbald compounds act selectively only against specific strains and the inhibition of fungal growth takes place only for the maximal concentration (MIC 1 mg/1 ml). The compounds disclosed in the PL214249 are conformationally variable, which diminishes their stability. Thus, in light of known prior art there is still a need for providing improved compounds showing broad-spectrum activity simultaneously against numerous bacterial strains, as well as fungal organisms, in minimal concentration, ensuring the creation of an effective medicament against infections caused by these microorganisms. A need identified in the prior art is also to provide compounds with improved stability while maintaining their effective cidal activity.

The object of the invention is therefore providing stable and biologically active substances used in medicine. The object has been realized by the present invention.

The object of the invention is a compound of a general formula:

wherein:

Rl denotes: N0 ₂ , CI or H,

R2 denotes: N0 ₂ or H,

R3 denotes: N0 ₂ , CI, CN, CH ₃ or H,

R4 denotes: N0 ₂ or H,

R5 denotes: N0 ₂ , CI or H,

R6 denotes: CH ₃ ,

R7 denotes: CH ₃ .

Preferably, the compound is selected from the group comprising: a) [(3,5-dimethyl-4H-l,2,4-triazol-4-yl)-amino](2-nitrophenyl)m ethanol (Formula 1); b) [(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino](3-nitrophenyl)me thanol (Formula 2); c) [(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino](4-nitrophenyl)me thanol (Formula 3); d) [(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino](2,4-dinitropheny l)methanol (Formula 4); e) [(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino](4-chloro-3-nitro phenyl)methanol (Formula 5); f) [(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino](4-methyl-3-nitro phenyl)methanol (Formula 6); g) [(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino](2-chloro-5-nitro phenyl)methanol (Formula 7). or a pharmaceutically acceptable salt thereof.

The object of the invention is also the new triazole derivative [(3,5-dimethyl-4H-l,2,4-triazol-4- yl)amino](2,4-dinitrophenyl)methanol (Formula 4) or a pharmaceutically acceptable salt thereof.

Preferably, the compound of the general formula described above is in a conformation referred to as extended.

A further object of the invention is a pharmaceutical agent containing the compound defined by the general formula above as an active principle, as well as particular embodiments thereof, or a pharmaceutically acceptable salt thereof.

Preferably, the agent is made in the form of an ointment, gel, tablet, capsule, powder, granules, syrup, injection or suppositories.

A further object of the invention is the compound defined by the general formula above, as well as particular embodiments thereof, or a pharmaceutically acceptable salt thereof for use as a medicament.

A further object of the invention is the compound defined by the general formula above, as well as particular embodiments thereof, or a pharmaceutically acceptable salt thereof for preventing and/or treating infections caused by bacteria and fungi.

Preferably, for preventing and/or treating fungal infections, particularly caused by Candida albicans, the compounds of formula 2, 3, 4, 6, 7 are used.

Preferably, for preventing and/or treating infections caused by Bacillus subtilis, Staphylococcus aureus and Proteus the compounds of formula 3 or 5 are used.

Preferably, the compounds are used in concentrations of 500 and 1000 μg/ml.

Preferably, for preventing and/or treating infections caused by Staphylococcus aureus and E. coli the compound of formula 7 is used. Preferably, the compound is used in concentrations of 250 μ^ιηΐ for S. aureus and 125 μ§/πι1 for E. coli respectively.

A further object of the invention is a method of preparation of the new triazole derivatives according to the invention, wherein a benzaldehyde derivative is reacted with 4-amino-3,5- dimethyl-4H-l,2,4-triazole, with equimolar amounts of the two substances dissolved in a solvent, followed by separation of the obtained compounds.

Preferably, the solvent used in the reaction of preparation of the new triazole derivatives is an organic solvent, such as methanol, ethanol, acetonitrile, propanol, isopropanol, DMF, DMSO, butanol, acetone, phenol, diethyl ether, chloroform, n-hexane and cyclohexane.

Preferably, the solvent is diethyl ether, chloroform, n-hexane and cyclohexane, more preferably n-hexane.

The new derivatives are compounds obtained as a result of reacting 4-amino-3,5-dimethyl-4H- 1,2,4-triazole with benzaldehyde derivative. They belong to a group of hemiaminals, known as considerably unstable and difficult to separate intermediate compounds in the Schiff bases synthesis. Preferably they are characterized by having two chiral centers and specifically selected substituents. Due to their properties they may be employed as active principles in prophylactic medicaments and those used in treating infections caused by the presence of bacteria and fungi.

It was unexpectedly found that introducing methyl groups to the triazole ring of hemiaminals influences their conformational stability while maintaining broad biological activity of the compounds in eliminating microorganisms. Among the new compounds no conformational variability of hemiaminals containing two methyl groups linked to a triazole ring was observed, in contrast to what was observed with these groups absent. Hemiaminals known from the prior art showed a tendency to adopt two conformations - extended and bent. The presently used methyl substituents prevent from adopting the bent conformation. This increases the diversity of benzaldehyde deivatives possible for use for medical purposes while simultaneously maintaining one, extended conformation, as well as beneficial biological activity of the hemiaminals.

The new compounds according to the invention may be used for the preparation of a medicament intended for prevention or treating infections caused by bacteria and fungi.

In order to better illustrate the invention, the description is supplemented with figures. Figure 1 shows the activity of the compounds against Bacilus subtilis bacteria. Designations: line with "x" - formula 3, line with squares - formula 4, line with triangles - formula 5.

Figure 2 shows the activity of the compounds against Staphylococcus aureus bacteria. Designations: line with squares - formula 1, line with stars - formula 3, line with triangles - formula 4, line with diamonds - formula 5, line with x - formula 7.

Figure 3 shows the activity of the compounds against Escherichia coli bacteria. Designations: line with squares - formula 4, line with triangles - formula 7.

Figure 4 shows the activity of the compounds against Proteus sp. bacteria. Designations: line with x - formula 3, line with squares - formula 4, line with triangles - formula 5.

Additionally, the present invention is illustrated with the following examples

Example 1. Chiral triazole compounds.

By reacting benzaldehyde derivatives (2-chlorobenzaldehyde, 4-cyanobenzaldehyde, 2- nitrobenzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, 2,4-dinitrobenzaldehyde, 4- chloro-3-nitrobenzaldehyde, 4-methyl-3-nitrobenzaldehyde, 2-chloro-5-nitrobenzaldehyde) with 4-amino-3,5-dimethyl-4H-l,2,4-triazole new compounds are obtained with chiral centers at the carbon atom and nitrogen atom linking the two rings.

Example 2. Synthesis of chiral triazole derivatives.

2.1. General principles of the synthesis

The course of the reaction of hemiaminals synthesis involves nucleophilic addition of an amine to the carbonyl group of the aldehyde. The presence of an acid in the reaction mix catalyzes separation of a water molecule, which results in the final end product being undesirable imines or, with large excess thereof - in inhibition of the reaction through blocking the amino group. The preferred reaction conditions are promoted by the absence of an acid in the reaction mix, adequate selection of the properties of donor-acceptor substituents in the phenyl ring and the specific properties of the amino residue. The latter is confirmed by the course of the synthesis of benzaldehyde derivatives and aniline leading to obtaining only a Schiff base. Appropriate selection of a solvent is significant to the course and efficiency of the reaction of hemiaminals synthesis. The synthesis is conducted in organic solvents, i.e. methanol, ethanol, acetonitrile, propanol, isopropanol, DMF, DMSO, butanol, acetone, phenol, diethyl ether, chloroform, n- hexane or cyclohexane. The highest efficiency in obtaining hemiaminals was achieved for the reaction conducted in n- hexane. The results achieved for the reaction conducted in cyclohexane, DMSO (dimethyl sulfoxide) and chloroform are also satisfactory. With regards to selectivity n-hexane as a solvent is comparable to acetonitrile.

The reagents were heated and mixed under reflux in 50°C for 9 hours. Then the reaction mix was washed with two small portions of acetonitrile and methanol and air-dried.

2.2. Synthesis of selected derivatives.

Synthesis of [(3.5-dimethyl-4H-1.2.4-triazol-4-yl)-amino](2-nitrophenyl)m ethanol (Formula 1).

4-amino-3,5-dimethyl-4H-l,2,4-triazole in quantity of 3.44xl0 ^"4 mole was dissolved in n-hexane at room temperature. In a separate vessel 3.44xl0 ^"4 mole of 2-nitrobenzaldehyde was dissolved in the same solvent at room temperature. Both solutions were poured into a round-bottomed flask. Total volume did not exceed 5 ml. The reagents were heated and mixed under reflux in 50°C for 9 hours. After cooling the obtained solution was left in a crystallization vessel, for the crystallization of the studied compound through solvent evaporation. After the crystallization process the obtained compound was filtered through a Biichner funnel at a reduced pressure and washed.

Synthesis of (3-nitrophenyl)[ 3.5-dimethyl-4H-1.2.4-triazol-4-yl)amino]methanol (Formula 2).

4-amino-3,5-dimethyl-4H-l,2,4-triazole in quantity of 3.44xl0 ^"4 mole was dissolved in acetonitrile at room temperature. In a separate vessel 3.44x1ο ^"4 mole of 3-nitrobenzaldehyde was dissolved in the same solvent at room temperature. Both solutions were poured into a round- bottomed flask. Total volume did not exceed 5 ml. The reagents were heated and mixed under reflux in 50°C for 9 hours. After cooling the obtained solution was left in a crystallization vessel, for the crystallization of the studied compound through solvent evaporation. After the crystallization process the obtained compound was filtered through a Biichner funnel at a reduced pressure and washed.

Synthesis of (4-nitrophenyl)[(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino]me thanol (Formula 3).

4-amino-3,5-dimethyl-4H-l,2,4-triazole in quantity of 3.44xl0 ^"4 mole was dissolved in n-hexane at room temperature. In a separate vessel 3.44xl0 ^"4 mole of 4-nitrobenzaldehyde was dissolved in the same solvent at room temperature. Both solutions were poured into a round-bottomed flask. Total volume did not exceed 5 ml. The reagents were heated and mixed under reflux in 50°C for 9 hours. After cooling the obtained solution was left in a crystallization vessel, for the crystallization of the studied compound through solvent evaporation. After the crystallization process the obtained compound was filtered through a Biichner funnel at a reduced pressure and washed.

Synthesis of f2,4-dinitrophenyl f3.5-dimethyl-4H-1.2.4-triazol-4-yl¼mino]methanol (Formula

4-amino-3,5-dimethyl-4H-l,2,4-triazole in quantity of 3.44xl0 ^"4 mole was dissolved in acetonitrile at room temperature. In a separate vessel 3.44xl0 ^'4 mole of 2,4-dinitrobenzaldehyde was dissolved in the same solvent at room temperature. Both solutions were poured into a round- bottomed flask. Total volume did not exceed 5 ml. The reagents were heated and mixed under reflux in 50°C for 9 hours. After cooling the obtained solution was left in a crystallization vessel, for the crystallization of the studied compound through solvent evaporation. After the crystallization process the obtained compound was filtered through a Biichner funnel at a reduced pressure and washed.

Synthesis of (4-chloro-3-nitrophenyl)[(3.5-dimethyl-4H-1.2.4-triazol-4-yl )amino] methanol (Formula 5).

4-amino-3,5-dimethyl-4H-l,2,4-triazole in quantity of 3.44xl0 ^"4 mole was dissolved in n-hexane at room temperature. In a separate vessel 3.44xl0 ^"4 mole of 4-chloro-3-nitrobenzaldehyde was dissolved in the same solvent at room temperature. Both solutions were poured into a round- bottomed flask. Total volume did not exceed 5 ml. The reagents were heated and mixed under reflux in 50°C for 9 hours. After cooling the obtained solution was left in a crystallization vessel, for the crystallization of the studied compound through solvent evaporation. After the crystallization process the obtained compound was filtered through a Biichner funnel at a reduced pressure and washed.

Synthesis of. (4-methyl-3-nitrophenyl)[(3.5-dimethyl-4H-1.2,4-triazol-4-yl )amino] methanol(Formula 6).

4-amino-3,5-dimethyl-4H-l,2,4-triazole in quantity of 3.44xl0 ^"4 mole was dissolved in acetonitrile at room temperature. In a separate vessel 3.44xl0 ^"4 mole of 4-methyl-3- nitrobenzaldehyde was dissolved in the same solvent at room temperature. Both solutions were poured into a round-bottomed flask. Total volume did not exceed 5 ml. The reagents were heated and mixed under reflux in 50°C for 9 hours. After cooling the obtained solution was left in a crystallization vessel, for the crystallization of the studied compound through solvent evaporation. After the crystallization process the obtained compound was filtered through a Biichner funnel at a reduced pressure and washed.

Synthesis of r2-chloro-5-nitrophenyl)[(3,5-dimethyl-4H-l _> 2.4-triazol-4-yl)amino] methanol (Formula 7).

4-amino-3,5-dimethyl-4H-l,2,4-triazole in quantity of 3.44xl0 ^"4 mole was dissolved in n-hexane at room temperature. In a separate vessel 3.44xl0 ^"4 mole of 2-chloro-5-nitrobenzaldehyde was dissolved in the same solvent at room temperature. Both solutions were poured into a round- bottomed flask. Total volume did not exceed 5 ml. The reagents were heated and mixed under reflux in 50°C for 9 hours. After cooling the obtained solution was left in a crystallization vessel, for the crystallization of the studied compound through solvent evaporation. After the crystallization process the obtained compound was filtered through a Biichner funnel at a reduced pressure and washed.

2.3. NMR spectroscopy.

The 1H NMR, ¹³ C NMR spectra as well as COSY and HMBC correlation spectra for characterization of the synthesized compounds were obtained with the following spectrometers: the Bruker Avance III 600 MHz spectrometer, the Bruker Avance 500 MHz spectrometer and the 300 MHz AMX Bruker NMR spectrometer. The studies were performed in the Nuclear Magnetic Resonance Laboratory of the Faculty of Chemistry, University of Wroclaw. The applied solvent was The signals in the NMR spectrum were calibrated on the solvent signal - 2.51 ppm in the 1H NMR spectrum and 39.91 ppm in the ¹³ C NMR spectra.

2.4. Mass spectrometry.

The ESI-MS spectra were obtained with the micrOTOF-Q-Bruker Dalonic device for the mass spectrum (m/z) 400-4000 in the Mass Spectrometry Laboratory of the Faculty of Chemistry, University of Wroclaw. The solvent was methanol and the ion source was electrospray (ESI).

2.5. Infrared spectroscopy.

The IR studies were performed with the Bruker 66/s FTIR (Bruker IFS66) spectrometer in the Infrared Spectroscopy Laboratory of the Faculty of Chemistry, University of Wroclaw. The measurements were performed in KBr. The spectra were measured in the 4000-400 cm ^"1 range.

2.6. Crystallography. The measurements for the monocrystals were performed with the Kuma KM4CCD diffractometer at the Faculty of Chemistry, University of Wroclaw. For the measurements in 100 K irradiation with a molybdenum lamp (ΜοΚ„, λ=0.71037 A) and a graphite monochromator were employed. Data reduction was performed with the CrysAlis software from Agilent Technologies. The structures were solved with direct methods and then refined using the Shelxs97 and Shelxl97 software.

2.7. Elemental analysis.

The analyses were performed with the Vario EL III CHNS element analyzer from Elementar at the Faculty of Chemistry, University of Wroclaw.

2.8. Compound identification.

[(3«5-dimethyl-4H-l _< 2.4-triazol-4-yl)-aminol(2-nitrophenvnmethanol (Formula 1)

Elemental analysis of the compound of formula C11H13 5O3:

NMR

1H (DMSO, 298 K, ppm, 300 MHz): 7.91 (m. 1H, H _A ); 7.89 (m, 1H, H _D ); 7.75 (m, 1H, H _c ); 7.60 (m, 1H, H _B ); 7.12 (d, lH, J _(C .H)-(N-H) = 8.12 Hz, N-H _F ); 6.92 (d, 1H, J _(C- H)-(O-H) = 5.33 Hz, 0-H _E ); 5.93 (dd, 1H, J(C-H)-(N-H) = 8.12 Hz, J _(C- H _H O-H) = 5.33 Hz, C-H _H ); 2.23 (s, 6H, CH _3(G) )

¹³ C[1H] (DMSO, 298 K, ppm, 300 MHz): 151.59 C ₃ , ₅ , 148.92 CM, 133.62 C ₉ , 133.37 Cn, 130.18 Cn, 129.07 Cio, 124.18 C ₁₃ , 79.23 C ₈ , 10.53 C _6>7

MS

ESI-MS (TOF, +M) Peak intensities for individual ions match the isotope distribution,

(CH ₃ OH) - m/z = 264.1 - [M+H] ⁺ 286.1 (100) - [M+Na] ⁺

302.1 - [M+K] ⁺

527.2 - [2M+H] ⁺ 549.2 - [2M+Na] ⁺

565.2 - [2M+K] ⁺

812.3 - [3M+Na] ⁺ 1075.4 - [4M+Na] ⁺

(CH ₃ CN) - m/z = 264.1 - [M+H] ⁺

367.1 - [M+Na-H+2CH ₃ CN] ⁺ 527.2 - [2M+H] ⁺

589.1 (100) - [(2M-H)+Na+CH ₃ CN] ⁺ IR (abbreviations: vw - very weak, w - weak, m - medium, s - strong, vs - very strong): KBr, 1. wavenumber [cm ^'1 ] 373 - w; 393 - w; 413 - w; 501 - m; 512 - w; 572 - m; 587 - vs; 607 - m; 624 - m; 663 - s; 685 - m; 719 - vs; 751 - s; 785 - vs; 858 - s; 874 - m; 892 - m; 959 - w; 978 - w; 1025 - m; 1040 - s; 1061 - vs; 1101 - vs; 1144 - w; 1164 - m; 1193 - s; 1248 - m; 1314 - m; 1361 - vs; 1417 - vs; 1445 - s; 1474 - m; 1498 - s; 1531 - vs; 1566 - s; 1612 - m; 1832 - vw; 1948

- vw; 1979 - vw; 2361 - vw; 2882 - s; 3114 - s; 3308 - vs; 3429 - s. nujol, 1. wavenumber [cm ^"1 ] 392 - w; 412 - vw; 480 - vw; 501 - w; 511 - w; 572 - w; 587 - m; 607 - w; 624 - w; 663 - m; 686 - w; 719 - s; 751 - m; 762 - m; 785 - m; 858 - m; 874 - w; 892 - w; 959 - w; 991 - w; 1025 - w; 1061 - s; 1101 - s; 1144 - w; 1164 - w; 1193 - m; 1248 - m; 1259

- m; 1315 - m; 1362 - vs; 1377 - s; 1418 - s; 1466 - s; 1498 - s; 1531 - vs; 1565 - m; 1612 - w; 1649 - w; 2855 - vs; 2925 - vs; 3151 - s; 3241 - s; 3308 - vs.

Crystallography

f(3,5-dimethyl-4 - ^r -1.2,4-triazol-4-vI)aminol(3-nitrophenvnmethanol (Formula 2)

Elemental analysis of the compound of a molecular formula C11H13N5O3:

NMR

1H (DMSO, 298 K, ppm, 300 MHz) 8.38 (s, IH, H _A ); 8.21 (dd, IH, J _B . _C = 8.09 Hz, ; J _B-D = 1.62 Hz, H _B ); 7.98 (d, IH, J _D-C = 7.98 Hz, H _D ); 7.69 (t, IH, J _C - _D = 7.98 Hz, ¾); 7.14 (d, IH, J _(C - _H) - ₍ N- H) = 7.17 Hz, N-H _F ); 6.75 (d, IH, J ₍ C-H)-(O-H) = 5.78 Hz, 0-H _E ); 5.57 (t, IH, J<C.HKO-HMN-H) = 6.47 Hz, C-HG); 2.31 (s, 6H, CU _m ).

¹³ C[1H] (DMSO, 298 K, ppm, 75 MHz) 151.50 C ₃₎₅ , 148.05 Ci ₃ , 142.99 C ₉ , 134.20 Cio, 130.24 C _u , 123.62 Ci2, 121.82 C _M , 83.22 C ₈ , 10.73 C ₆₍₇ .

IR (abbreviations: vw - very weak, w - weak, m - medium, s - strong, vs - very strong):

KBr, 1. wavenumber [cm ^"1 ] 503 - w; 583 - w; 608 - w; 629 - w; 679 - s; 693 - w; 730 - m; 762 - w; 803 - m; 866 - w; 907 - w; 917 - w; 944 - w; 1003 - w; 1050 - s; 1093 - m; 1202 - m; 1248 - w; 1353 - vs; 1379 - m; 1421 - s; 1527 - vs; 1563 - m; 1585 - w; 1617 - w; 1648 - w; 1704 - w; 2931 - m; 3115 - s; 3253 - s; 3312 - s.

MS

ESI-MS (TOF, +M) Peak intensities for individual ions match the isotope distribution, M CiiHi ₃ N ₅ 0 ₃ = 263.2640 gmol

(CH ₃ OH) - m/z = 264.1 [M+H] ⁺ (100%)

286.1 [M+Na] ⁺

302.1 [M+K] ⁺

549.2 [2M+Na] ⁺ (CH ₃ CN) - m/z = 264.1 [M+H] ⁺ (100%) i(3,5-dimethyl-4H-l,2,4-triazol-4-vnaminol(4-nitrophenvnmeth anol fFormula 3)

Elemental analysis of the compound of a molecular formula CnH ₁₃ N ₅ 0 ₃ :

%C %H %N

calculated 50.18 4.98 26.61

measured 50.36 4.03 25.50 NMR

1H (DMSO, 298 K, ppm, 300 MHz) 8.25 (d, 2H, J _B-A = 8.65 Hz, H _B ); 7.80 (d, 2H, J _A-B = 8.65 Hz, H _A ); 7.13 (d, 1H, J( _C .H N-H) = 7.18 Hz, N-H _E ); 6.73 (d, 1H, J _(C -H)-(O-H) = 5.67 Hz, 0-H _D ); 5.55 (t, 1H, J(C.H)-(O-H),(N.H) = 6.42 Hz, C-H _c ); 2.23 (s, 6H, CH ₃₍ F)).

¹³ C[1H] (DMSO, 298 K, ppm, 75 MHz) 151.53 C ₃ , ₅ , 147.91 C ₁₂ , 131.09 C ₉ , 128.67 do, 123.72 Cii, 83.42 C ₈ , 10.73 C ₆ , ₇ .

IR (abbreviations: vw - very weak, w - weak, m - medium, s - strong, vs - very strong):

KBr, 1. wavenumber [cm ¹ ] 385 - m; 477 - m; 509 - m; 528 - w; 592 - s; 628 - w; 656 - m; 678

- m; 693 - s; 718 - s; 752 - s; 766 - m; 789 - vs; 857 - s; 896 - s; 941 - vw; 980 - w; 1015 - s; 1064 - vs; 1106 - s; 1119 - w; 1191 - s; 1250 - s; 1273 - s; 1350 - vs; 1384 - s; 1419 - s; 1463

- s; 1511 - vs; 1569 - vs; 1600 - s; 1607 - s; 1684 - m; 1705 - m; 1811 - vw; 1944 - vw; 2361

- m; 2705 - s; 2854 - vs; 2914 - vs; 3079 - vs; 3304 - vs; 3431 - s. Nujol, 1. wavenumber [cm ¹ ] 385 - m; 477 - m; 509 - w; 529 - vw; 592 - s; 628 - w; 656 - m; 678 - m; 693 - s; 720 - vs; 752 - s; 766 - s; 789 - vs; 857 - vs; 896 - vs; 978 - m; 1014 - s; 1064 - vs; 1106 - s; 1119 - w; 1191 - s; 1250 - s; 1273 - s; 1350 - vs; 1378 - vs; 1419 - s; 1464 - vs; 1511 - vs; 1569 - s; 1599 - w; 1607 - w; 1681 - w; 1705 - vw; 2701 - w; 2925 - m; 3303 - m.

MS

ESI-MS (TOF, +M) Peak intensities for individual ions match the isotope distribution, M CiiHi ₃ N ₅ 0 ₃ = 263.2640 g/mol

(CH ₃ OH) - m/z = 264.1 [M+H] ⁺ (100%)

286.1 [M+Na] ⁺

302.1 [M+K] ⁺

[(3.5-dimethyl-4H-l,2.4-triazol-4-vnaminol(2.4-dinitror&g t;henvnmethanol fFormula 4)

Elemental analysis of the compound of a molecular formula CnH^N^:

NMR

1H (DMSO, 298 K, ppm, 300 MHz) 8.71 (d, 1H, J _C -B = 2.30 Hz, H _c ); 8.57 (dd, 1H, J _B-A = 8.77 Hz, J _B .c = 2.30 Hz, H _B ); 8.16 (d, 1H, J _A-B = 8.77 Hz, H _A ); 7.28 (d, 1H, J _(C -HHN-H) = 8.35 Hz, N- H _F ); 7.21 (d, 1H, J _(C .H>(O-H) = 5.01 Hz, 0-H _E ); 5.95 (dd, 1H, J _(C- H _W N.H) = 8.35 Hz, J _(C .H _H O-H) = 5.01 Hz, C-HD); 2.23 (s, 6H, CH _3(G )).

¹³ C[1H] (DMSO, 298 K, ppm, 75 MHz) 151.53 C _3>5 , 148.74 Ci ₄ , 147.94 C12, 139.66 C ₉ , 131.04 C11, 127.61 Cio, 119.80 C13, 79.13 C ₈ , 10.56 C _6;7 .

IR (abbreviations: vw - very weak, w - weak, m - medium, s - strong, vs - very strong):

KBr, 1. wavenumber [cm ^"1 ] 376 - vw; 463 - vw; 502 - w; 588 - w; 649 - vw; 663 - w; 678 - vw; 712 - m; 727 - w; 743 - w; 760 - w; 766 - w; 790 - m; 835 - m; 856 - vw; 889 - w; 905 - m; 922 - vw; 977 - vw; 1022 - w; 1059 - m; 1083 - m; 1127 - w; 1147 - vw; 1191 - w; 1247 - w; 1295 - w; 1350 - vs; 1415 - m; 1469 - w; 1506 - m; 1537 - vs; 1566 - m; 1608 - m; 2361 - vw; 2744 - m; 2879 - m; 3106 - m; 3305 - s; 3431 - w.

Nujol, 1. wavenumber [cm ^"1 ] 377 - vw; 464 - m; 502 - s; 588 - vs; 649 - w; 663 - s; 679 - w; 712 - vs; 727 - vs; 743 - s; 759 - s; 766 - s; 790 - s; 814 - m; 835 - vs; 856 - m; 889 - s; 905 - vs; 923 - w; 977 - w; 1022 - m; 1041 - s; 1059 - vs; 1084 - vs; 1127 - m; 1146 - m; 1191 - s; 1247 - m; 1295 - m; 1315 - s; 1366 - vs; 1415 - s; 1464 - vs; 1538 - vs; 1567 - s; 1607 - s; 1710 - vw; 1973 - vw; 2725 - m; 2927 - s; 3104 - m; 3304 - m; 3655 - vw.

MS

ESI-MS (TOF, +M) Peak intensities for individual ions match the isotope distribution, M CnHi ₂ N ₆ 0 ₅ = 308.2660 g/mol.

(CH ₃ OH) - m/z = 309.1 [M+H] ⁺ (100)

331.1 [M+Na] ⁺ 347.0 [M+K] ⁺

(CH ₃ CN) - m/z = 309.1 [M+H] ⁺ (100)

679.1 [2M+Na+CH ₃ CN] ⁺ Cr stallography

f(3,5-dimethyl-4H-l,2,4-triazol-4-yl¼minoK4-chloro-3-nitrop henyl)methanol (Formula 5)

Elemental analysis of the compound of a molecular formula CiiHi ₂ Ns0 ₃ Cl:

NMR

^J H (DMSO, 298 K, ppm, 300 MHz) 8.18 (d, 1H, J _A-C = 1.72 Hz, H _A ); 7.84 (d, 1H, J _C . _A = 1.72 Hz, He); 7.82 (m, 1H, H _B ); 7.15 (d, 1H, J _(C .HMN-H) = 7.44 Hz, N-H _F ); 6.81 (d, 1H, J _(C- HHO.H) = 5.72 Hz, 0-H _E ); 5.52 (t, 1H, J(C-H)-(O-H) ₎₍ N-H) = 6.58 Hz, C-H _D ); 2.31 (s, 6H, CH ₃₍ G)).

¹³ C[1H] (DMSO, 298 K, ppm, 75 MHz) 151.47 C _3>5 , 147.77 C ₁₃ , 141.81 C ₉ , 132.80 Cio, 131.91 Cu, 125.00 C ₁₂ , 124.18 C ₁₄ , 82.71 C ₈ , 10.72 C _6>7 .

IR (abbreviations: vw - very weak, w - weak, m - medium, s - strong, vs - very strong):

KBr, 1. wavenumber [cm ^"1 ] 411 - vw; 487 - w; 505 - w; 586 - w; 603 - w; 633 - w; 664 - w; 680 - w; 691 - w; 744 - w; 763 - w; 809 - m; 863 - m; 917 - vw; 947 - w; 980 - vw; 1025 - m; 1048 - s; 1091 - w; 1138 - w; 1154 - vw; 1204 - w; 1249 - w; 1367 - s; 1423 - m; 1479 - w;

1502 - m; 1532 - vs; 1566 - m; 1607 - w; 1702 - w; 2729 - w; 2878 - m; 3105 - m; 3261 - s; 3430 - m.

Nujol, 1. wavenumber [cm ] 411 - w; 487 - s; 505 - s; 515 - w; 586 - m; 603 - m; 633 - w; 664 - s; 680 - m; 691 - m; 743 - s; 763 - s; 809 - vs; 863 - s; 917 - w; 947 - s; 980 - m; 1025 - s; 1048 - vs; 1091 - m; 1138 - m; 1153 - m; 1204 - s; 1249 - m; 1377 - vs; 1424 - s; 1464 - vs;

1503 - s; 1532 - vs; 1566 - s; 1606 - w; 1670 - w; 1700 - vw; 2727 - m; 2924 - s; 3259 - m.

MS

ESI-MS (TOF, +M) Peak intensities for individual ions match the isotope distribution, M CnHaz sOsCl = 297.7060 g/mol.

(CH ₃ OH) - m/z = 298.1 [M+H] ⁺

320.0 [M+Na] ⁺ (100%)

336.0 [M+K] ⁺

[(3,5-dimethyl-4H-1.2,4-triazol-4-yl)aniinol 4-methyl-3-nitroDhenyl)methanol fFormula 6)

Elemental analysis of the compound of a molecular formula ^ ₂ Η ₁₅ Ν ₅ θ ₃ : %C %H %N

calculated 51.98 5.45 25.26

measured 51.99 4.97 24.88

NMR

1H (DMSO, 298 K, ppm, 300 MHz) 8.11 (d, IH, J _A-C = 1.18 Hz, H _A ); 7.76 (dd, IH, J _C . _B = 7.82 Hz, JC-A = 1.42 Hz, He); 7.52 (d, IH, J _B-C = 7.82 Hz, H _B ); 7.07 (d, IH, J _(C -H)-(N-H) = 6.87 Hz, N- H _G ); 6.65 (d, IH, J _(C -H).(0-H) = 5.69 Hz, 0-H _F ); 5.49 (t, IH, J ₍ C-H)-(O-H) ₎₍ N-H) = 6.40 Hz, C-H _E ); 2.50 (s, 3H, CH ₃₍ D)); 2.30 (s, 6H, CH _3(H) ).

15

CH ₃

10 .14

-CH

6 ^'

\\ //

— (DMSO, 298 K, ppm, 75 MHz) 151.00 C ₃ , ₅ , 148.60 Ci ₃ , 139.94 C ₉ ,i ₂ 132.61 Cu, 131.71 Cio, 122.37 Cu, 82.62 C ₈ , 19.25 Ci ₅ , 10.23 C _6>7 .

IR (abbreviations: vw - very weak, w - weak, m - medium, s - strong, vs - very strong): KBr, 1. wavenumber [cm ¹ ] 429 - vw; 504 - w; 540 - vw; 569 - m; 615 - w; 666 - w; 682 - m; 731 - m; 754 - m; 762 - w; 777 - m; 810 - m; 846 - vw; 862 - m; 912 - w; 952 - w; 978 - vw; 1024 - w; 1065 - s; 1080 - s; 1165 - vw; 1198 - m; 1246 - w; 1331 - vs; 1346 - vs; 1382 - m; 1419 - m; 1454 - m; 1500 - s; 1524 - vs; 1564 - m; 1572 - s; 1623 - w; 1701 - vw; 1820 - vw; 2361 - w; 2723 - m; 2873 - m; 2924 - m; 2991 - m; 3105 - s; 3309 - vs; 3430 - m.

Nujol, 1. wavenumber [ran ^"1 ] 429 - w; 504 - m; 539 - w; 569 - s; 615 - s; 667 - s; 682 - s; 730 - vs; 753 - s; 762 - s; 777 - s; 810 - s; 846 - m; 862 - s; 912 - m; 952 - m; 977 - m; 1064 - vs; 1079 - s; 1165 - m; 1198 - s; 1245 - m; 1331 - vs; 1378 - vs; 1464 - vs; 1489 - s; 1524 - vs; 1564 - m; 1572 - m; 1622 - vw; 1699 - vw; 2915 - s; 3308 - w.

MS

ESI-MS (TOF, +M) Peak intensities for individual ions match the isotope distribution, M Ci ₂ H ₁₅ N ₅ 0 ₃ = 277.2900 g/mol.

(CH ₃ OH) - m/z = 278.1 [M+H] ⁺ (100 %)

300.1 [M+Na] ⁺

316.1 [M+K] ⁺ (CH ₃ CN) - m/z = 278.1 [M+H] ⁺ (100)

K3,5-dimethyl-4H-l,2,4-triazol-4-vUaminoK2-chloro-5-nitro phenvnmethanol (Formula 7)

Elemental analysis of the compound of a molecular formula C11H12N5O3CI:

NMR

1H (DMSO, 298 K, ppm, 300 MHz) 8.44 (d, 1H, J _A-B = 2.71 Hz, H _A ); 8.22 (dd, 1H, J _B-C = 8.80 Hz, J _B-A = 2.71 Hz, H _B ); 7.78 (d, 1H, J _C . _B = 8.80 Hz, H _c ); 7.21 (d, 1H, J _(C- H)-(N-H) = 7.11 Hz, N- H _F ); 6.98 (d, 1H, = 5.49 Hz, 0-H _E ); 5.72 (dd, 1H, J _(C .HHN-H) = 6.98 Hz, J ₍ C-H) _-( O-H) = 5.49 Hz, C-H _D ); 2.31 (s, 6H, CH _3(G )).

IR (abbreviations: vw - very weak, w - weak, m - medium, s - strong, vs - very strong):

KBr, 1. wavenumber [cm ^"1 ] 430 - vw; 465 - w; 502 - w; 511 - w; 528 - m; 570 - w; 585 - m; 612 - m; 630 - w; 662 - m; 682 - w; 692 - w; 745 - s; 769 - w; 800 - m; 842 - m; 858 - w; 913 - m; 950 - w; 985 - w; 1027 - m; 1041 - s; 1068 - m; 1103 - m; 1143 - vw; 1196 - m; 1247 - m; 1278 - m; 1351 - vs; 1377 - m; 1419 - s; 1462 - m; 1517 - vs; 1550 - m; 1576 - m; 1610 - m; 1701 - w; 1825 - vw; 2858 - m; 3070 - s; 3099 - s; 3309 - s; 3430 - m.

MS

ESI-MS (TOF, +M) Peak intensities for individual ions match the isotope distribution, M CiiHi ₂ N ₅ 0 ₃ Cl = 297.7060 g mol.

(CH ₃ OH) - m/z = 298.1 [M+H] ⁺

320.0 [M+Na] ⁺ (100%)

336.0 [M+K] ⁺ Example 3. Biological properties of the new compounds

Bactericidal activity of the seven newly synthesized chiral triazole derivative compounds was determined.

The Microplate Alamar Blue® Assay (MABA) was conducted for a series of concentrations of the tested compounds. To the so prepared material bacterial suspension of a determined optical density was added and 24 hour incubation was conducted during which the bacteria are exposed to a potentially bactericidal chemical compound. Subsequently the Alamar Blue® dye is added, which distinguishes between living bacteria having metabolic activity and dead ones. The activity was measured with a fluorimeter.

The Minimal Inhibitory Concentration (MIC) was determined by calculating the inhibition of bacterial growth after plotting inhibition curves.

Negative 'no growth' and positive 'growth' controls were prepared.

Tested compounds

Chiral triazole derivatives were tested - the compounds numbered according to the patent application: formula 1, formula 2, formula 3, formula 4, formula 5, formula 6, formula 7. Each compound was tested in triplicates.

Bacterial strains

In the studies bacterial strains from The Polish Collection of Microorganisms (PMC) at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wroclaw were used: Staphylococcus aureus PCM 2602, Escherichia coli PCM 1144, Pseudomonas aeruginosa PCM 2058, Candida albicans PCM 2560, Proteus sp. PCM 542, Bacillus subtilis, Enterococcus hirae PCM 2559.

Reagents and materials for the MABA test a) Alamar Blue® dye (Biosource, USA) - marker for bacteria for fluorescence measurements, b) dimethyl sulfoxide (DMSO, Merck) - solvent for the tested compounds, c) sterile PBS. Bacterial culture medium

All microorganisms were cultured in standard nutrient broth (Difco). Bacterial culture

Bacteria, stored on agar slants (inoculum - 3 ^rd passage), were each time seeded to a liquid medium and cultured for 24 hours in 37°C. Optical density measurement of bacterial suspension was performed for the wavelength 550 nm. In order to standardize the measurement the suspension was diluted as needed to absorbance of 0.5. Preparation of compounds for determination of bactericidal activity using the MABA test

The compounds in test portions of 10 mg were dissolved in 100 μΐ DMSO and then diluted to the volume of 10 ml in sterile PBS (1 mg/ml).

The tested compounds were diluted in an exponential series in the liquid bacterial medium on a sterile 96-well plate for cell culture (Nunc), the first dilution being 1 mg/1 ml, the next 0.5 mg/1 ml, 0.25 mg/1 ml etc. For this purpose 100 μΐ of sterile PBS was added to each well (excluding the first), followed by addition of 200 μΐ of the tested compound to the first well in line. 100 μΐ of each was taken and transferred (diluting it) to the consecutive wells.

The controls for the test were:

- sterile PBS (200 μΐ),

- PBS (100 μΐ) + tested compounds 1 mg/ml (100 μΐ);

- PBS (100 μΐ) + bacterial culture (100 μΐ).

Bacterial suspension was added to the plate with serial dilutions of the tested compounds and incubated in 37°C for 24 hours. The bacterial cell - tested compound complex was then stained with the Alamar Blue® dye (20 μΐ) and incubated for 2 hours. The fluorescence level, the source of which was the dye absorbed by the remaining living bacterial cells, was determined using the EnSpire 2300 reader (Perkin Elmer) for the wavelength 570 - 590 nm (excitation - emission).

Biological properties of the obtained compounds

Bactericidal activity

The bacterial growth inhibitory activity of the compounds was determined using MIC (Minimal Inhibitory Concentration), being the minimal concentration [^g/ml] exhibiting bacterial growth inhibition.

Table 1. Summary of the results

Bactericidal/fungicidal activity of the tested compounds

Inhibition of growth for some of the studied bacteria was observed. The compound of formula 4 shows the most effective activity against all studied microorganisms. The result demonstrates a substantial bactericidal potential, both against gram-positive and gram-negative bacteria.

The compounds defined by formula 2, formula 3, formula 4, formula 6 and formula 7 have fungicidal activity (Candida albicans) in concentrations of 500 and 1000 μg/ml. The compound defined by formula 7 also showed activity against Staphylococcus aureus (250 μg ml) and E. coli (125 μg/ml). The compounds defined by formula 3 and formula 5 yere active against Bacillus subtilis, Staphylococcus aureus and Proteus sp. in concentrations of 500 and 1000 μg/ml.

Formula 1

[(3,5-dimethyl-4H-l,2,4-triazol-4-yl)-amino](2-nitrophenyl)m ethanol

Formula 2

[(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino](3-nitrophenyl)me thanol

Formula 3

[(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino](4-nitrophenyl)me thanol

Formula 4

[(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino](2,4-dinitropheny l)methanol

Formula 5

[(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino](4-chloro-3-nitro phenyl)methanol

Formula 6

[(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino](4-methyl-3-nitro phenyl)methanol

Formula 7

[(3,5-dimethyl-4H-l,2,4-triazol-4-yl)amino](2-chloro-5-ni trophenyl)methanol