Trіazolo[1,5-c]quіnazolіnes. Synthesis, antimicrobial and antifungal activity [quinazolin-4(3H)-ylidene]hydrazides of carboxylic acids (Message 1)

The modern standards and algorithms of antimicrobial therapy, that ensure a high level of the treatment’s quality and prevention of the most infectious-inflammatory diseases, provide wide using of chemotherapeutic agents. However, the modern medicines do not always have satisfactory chemotherapeutic and pharmacological properties; this fact is most often associated with the resistance of microorganisms to them. That is why the creation of the new chemotherapeutic drugs by natural antibiotics’ chemical and microbiological modification, the chemical synthesis of the new substances among the various classes of organic compounds is a topical problem.

The modern standards and algorithms of antimicrobial therapy, that ensure a high level of the treatment's quality and prevention of the most infectious-inflammatory diseases, provide the wide using of chemotherapeutic agents.
However, the modern medicines do not always have satisfactory chemotherapeutic and pharmacological properties; this fact is most often associated with the resistance of microorganisms to them.
That is why the creation of the new chemotherapeutic drugs by natural antibiotics' chemical and microbiological modification, the chemical synthesis of the new substances among the various classes of organic compounds is a topical problem [1].
The quinazoline derivatives belong to one of the most perspective types of heterocyclic compounds that are used in modern pharmaceutical practice.
Sufficiently ample opportunity of the quinazoline cycle's chemical modification creates the significant prerequisites for the design of the new potential medical drugs [2].The results of the previous studies [3], showed that some (3H-quinazolin-4-ylidene)hydrazides of carboxylic acids have the expressed antimicrobial, antifungal and other activities.
This fact substantiates the reasonability of the further researches aimed at the creation of new antimicrobial medical drugs with the greater efficiency and less toxicity in comparison with the available on the pharmaceutical market drugs.
Moreover, hydrazides of carboxylic acids are an interesting group of the organic compounds [4].This group due to the combination of two reaction centers in the molecule is widely used in cyclocondensation reactions for the heterocyclic systems' forming [5].

The aim
The aim of the study is the developing of the simple and accessible methods for the synthesis of new [quinazoline-4(3H)-ylidene]hydrazides of cycloalkyl-(hetaryl) carboxylic acids, analysis of their physical-chemical properties, antimicrobial and antifungal activity.

Materials and methods
The experimental chemical part.Melting points were determined in open capillary tubes and were uncorrected.The elemental analyses (C, H, N, S) were performed using the ELEMENTAR vario EL Cube analyzer (USA).Analyses were indicated by the symbols of the elements or functions within ±0.3 % of the theoretical values.The 1 H NMR spectra (400 MHz) and 13 C NMR spectra (100 MHz) were recorded on a Varian-Mercury 400 (Varian Inc., Palo Alto, CA, USA) spectrometer with TMS as the internal standard in DMSO-d6 solution.The LC-MS were recorded using a chromato-mass spectrometric system which consisted of a high-performance liquid chromatograph "Agilent 1100 Series" (Agilent, Palo Alto, CA, USA) equipped with a diode-matrix and mass-selective detector "Agilent LC/MSD SL" (atmospheric pressure chemical ionization -APCI).The purity of all obtained compounds was checked by Вопросы фармации (1.1-1.5) and 4-chloroquinazoline (2.1-2.5) was implemented by well-known methods, with constants that correspond to literature data [6].

Methods of synthesis of N'-(quinazolin-4(3H)-ylidene) carbohydrazides (3.1-3.25).
Method A. 1.95 g (11 mmol) of carbonyldiimidazole was added to the solution of 11 mmol of the corresponding carboxylic acid in 10 ml of anhydrous dioxane.The formed mixture was heated on a water bath at 60-80 °C for 1 hour, protecting from the air moisture, using tube filled by calcium chloride.Then 10 mmol of the corresponding substituted 4-hydrazinoquinazolines (1.1-1.5) were added to the reaction mixture with stirring.Then it was left for 8 hours at ambient temperature.The formed mixture was poured into water and acidified by acetic acid to pH 6-7.The formed precipitate was filtered off, dried, crystallized if necessary.
Method B. 11 mmol of the corresponding carboxylic acid hydrazide was added to the solution of 10 mmol of the corresponding substituted 4-chloroquinazoline (2.1-2.5) in 10 ml of dioxane; kept it on a water bath at 60 °C for 8 hours.After cooling, the reaction mixture was poured into water, alkalified to pH 6-7 by 5 sodium hydrocarbonate solute.The formed precipitate was filtered off, dried, crystallized if necessary.
The experimental biological part.The sensitivity of the microorganisms to the synthesized compounds was evaluated according the described methods [7].The assay was conducted on Mueller-Hinton medium by two-fold serial dilution of the compound in 1 ml, after that 0.1 ml of microbial seeding (106 cells/ml) was added.The minimal inhibitory concentration of the compound was determined by the absence of visual growth in the test tube with a minimal concentration of the substance, and then the minimal bactericide/fungicide concentration was determined by the absence of growth on agar after inoculation of the microorganism from the transparent test tubes.Dimethylsulfoxide was used as a solvent, with an initial solution concentration of 1 mg/ml.Preliminary screening was performed on Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Candida albicans ATCC 885-653 standard test cultures.Nitrofual, Trimetoprim and Ketoconazole were used as the reference compounds with proven antibacterial/antifungal activity.Additional quality control of the culture medium and solvents was conducted by commonly used methods [8,9].

Results and its discussion
Synthesis of (3H-quinazolin-4-ylidene)hydrazides cycloalkyl-(hetaryl) carboxylic acid 3.1-3.25 as the convenient precursors for the preparation of the corresponding tricyclic derivatives [10], was carried out in two alternative ways: by acylation of the corresponding substituted 4-hydrazinoquinazolines (1.1-1.5) by activated cycloalkyl-(hetaryl) carboxylic acids (Method A, scheme 1) and by interaction substituted 4-chloroquinazolines (2.1-2.5) with the hydrazides of the corresponding acids (Method B, scheme 1) [11,12].The acylation of the compounds 1.1-1.5 was realized by imidazolides of alicyclic or heterocyclic acids (Method A).It should be noted that the regioselectivity of the reaction is determined by the place of the primary nucleophilic attack, that may be represented by the final nitrogen atom of the hydrazine moiety or by the endocyclic N (1) and N (3) atoms of quinazoline.Later, the alternative synthesis of some N-acyl derivatives (3.11, 3.17) was carried out, it was based on the interaction of substituted 4-chloroquinazolines (2.1-2.5) with hydrazides of the corresponding alicyclic acids (Method B).The using of this approach allowed the definitely establishing of the reactions` direction.
Each of the mentioned approaches has both advantages and disadvantages.Among the characteristics of the carbonyldiimidazole method, it is necessary to note the rate of performing, high yields and purity of the final products, which, in our opinion, are significant advantages, despite of the necessity of anhydrous solvents' using.One of the features of this method is the possible one-stage heterocyclization of the corresponding hydrazides into triazoloquinazoline systems, provided by the extended time and the increased temperature regime of the reaction.But the chosen reaction conditions made possible to get the individual N'-[quinazoline-4(3H)-ylidene]-2-(3)-cycloalkyl(hetaryl)-hydrazides with the high yields.
Among the disadvantage of the second method, it is necessary to note the duration of hydrazides of alicyclic or heterocyclic acids' synthesis and also the using of shortlived substituted 4-chloroquinazolines (2.1-2.5) as initial compounds.
In the LC-MS spectra (APCI) of the compounds 3.1-3.25,the signals of positive ions [M+1] were recorded which confirms the expected molecular weight of the synthesized compounds.For compounds 3.11-3.16,the signals of positive ions [M+3] were additionally recorded, which characterizes the «isotopic profile» of sulfur [13].
1 Н-NMR-spectra were characterized by the appearance of a low-field proton singlet of the exocyclic NH-group of the quinazoline cycle at the 11.96-10.37ppm [11,12] and the amide NH-proton at the 11.03-9.90ppm, which in some cases (3.13, 3.18, 3.19, 3.22, 3.23) did not appear due to rapid deuterium exchange or doubles due to hydrazide hydrazone tautomerism (3.9, 3.12).It is necessary to note that it was not possible to describe the multiplicity and characteristic chemical shifts of the aromatic protons of both quinazoline residue and the heterocyclic component of the molecule for hydrazides 3.11, 3.13-3.16, 3.18-3.21, 3.23-3.25,since they were observed as multiplets, which was a consequence of prototrophic tautomerism and their significant solubility in DMSO.
In the 1 H NMR spectra of compounds 3.1-3.6,the signals of protons of the alicyclic residue were registered in a high field.In some cases mentioned signals were doubled, that was also caused by the tautomeric transitions.As for compounds 3.1 and 3.2 four protons of 2 nd and 3 rd position of the cyclopropyl moiety were registered as a multiplet at the 0.97-0.63ppm, and the proton of 1 st position as a multiplet at 1.67-1.84ppm.The splitting of the protons of the adamantan residue of compound 3.3 was interesting: they resonated as two six-proton doublets of «bridge» protons at the 1.75 ppm (H-4.4', H-6.6', H-10.10') and the 1.92 ppm (Н-2,2', Н-8,8', Н-9,9'), and three-proton singlet of «nodal» protons at the 2.06 ppm (Н-3, Н-5, Н-7); for compounds 3.5 and 3.6, the protons of the adamantan residue resonated as multiplets at 2.23-1.55ppm.In the 1 H NMR spectra of compounds 3.4, 3.7, 3.13, 3.18, there was a three-proton singlet of the methyl group of the position 8 at the 2.49-2.53ppm.
It is interesting to notice that the 1 Н-NMR spectrum of compound 3.17 made it possible to characterize both aromatic and heterocyclic protons.The aromatic protons of the quinazoline ring form the characteristic ABCD system: H-5 and H-8 were observed as doublets at the 8.00 and the 7.80 ppm respectively, and H-6 and H-7 -triplets at the 7.57 and the 7.50 ppm respectively [11,12,14].As for proton H-2 of quinazoline cycle, it resonated as a singlet at the 8.12 ppm, that is typical for most compounds of this series [2,15].Protons of heteryl substituents had classical chemical shifts and multiplicity [16,17].
As a result of the study of antimicrobial and antifungal activity, it was established (Table 1) that synthesized compounds in the concentration range of 200.0-12.5 μg/ml were active against the studied strains.The results' analysis of the effect of the test compounds on the growth of Staphylococcus aureus and Escherichia coli, showed that the greatest bacteriostatic (12.5-50.0μg/ml) and bacterici- Problems of pharmacy dal (50-100 μg/ml) effects were exhibited by substances 3.22 and 3.23, which in hydrazide fragment contain a pyridine residue (3.22, 3.23).Towards to Pseudomonas aeruginosa, the investigated substances did not exhibit significant bacteriostatic and bactericidal activity (MIC-50-100 μg/ml, MBС-100-200 μg/ml).

Conclusions
1. Preparative methods for the synthesis of [quinazoline-4(3H)-ylidene]hydrazides of cycloalkyl-(hetaryl) carboxylic acids are considered.The choice of presented methods depends on order of receiving of the condensed heterocyclic and biologically active compounds.
2. The individuality and structure of the synthesized compounds was confirmed by elemental analysis, 1 Н-NMR and LC-MS spectra.It was shown, that prototropic (hydrazine-hydrazone) tautomerism is typical for synthesized compounds and, importantly, this tautomeric process leads to the changes in the integrated intensity and chemical shifts of both aromatic protons and protons of alicyclic groups.
Prospects for further research.During the next stage of our studies, it is planned to establish acute toxicity indicators, as well as to study the biological activity of synthesized compounds.In addition, the obtained compounds are prospective synthons in order to receive the tricyclic derivatives.Вопросы фармации , 8.43 (s, 1H, H-2), 10.14 (s, 1H, NH), 11.40 (s, 1H, NH); LC-MS, m/z = 247 [M+1]; Anal.Calcd for C 12 H 11 FN 4 O: C Scheme 1