Design and Evaluation of Novel Antimicrobial and Anticancer Agents Among Tetrazolo[1,5-c]quinazoline-5-thione S-Derivatives

The novel heterocyclization of 5-(2-aminophenyl)-1H-tetrazole with potassium ethylxanthogenate or carbon disulfide was proposed. The potassium salt of the tetrazolo[1,5-c]quinazoline-5-thione was subsequently modified by alkylation with proper halogen derivatives to (tetrazolo[1,5-c]quinazolin-5-ylthio)alkyls, N,N-dialkylethylamines, 1-aryl-2-ethanones, 1-(alkyl)aryl-2-ethanols, carboxylic acids, and esters. The structures of all newly synthesized compounds were confirmed by FT-IR, UV-vis, LC-MS, 1H, 13C NMR, and elemental analysis data. The substances were screened for antibacterial and antifungal activities (100 μg) against Escherichia coli, Staphylococcus aureus, Enterobacter aerogenes, Entrococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Candida albicans. Preliminary bioluminescence inhibition tests against Photobacterium leiognathi Sh1 showed that substances 5.2–5.4, 6.1, 7.1 with ethanone or carboxylic acid substituents showed toxicity against bacteria cells. The substances chosen by the US National Cancer Institute (NCI) were screened for their ability to inhibit 60 different human tumor cell lines, where 2-(tetrazolo[1,5-c]quinazolin-5-ylthio)-1-(4-tolyl)ethanone (5.2), 3-(tetrazolo[1,5-c]quinazolin-5-ylthio)propanoic and related 3-metyl-butanoic acids (6.2, 6.3), and ethyl tetrazolo[1,5-c]quinazolin-5-ylthio)acetate (7.2) showed lethal antitumor activity (1.0 μM) against the acute lymphoblastic leukemia cell line (CCRF-CEM), and substances 5.2 and 6.3 exhibited moderate anticancer properties inhibiting growth of the leukemia MOLT-4 and HL06-(TB) cell lines. The moderate antitumor activity was demonstrated in 1-(2,5-dimethoxyphenyl)-2-(tetrazolo[1,5-c]quinazolin-5-ylthio)ethanone (5.4) against the CNS cancer cell line SNB-75. Comparing the docking mode of the Gefitinib and synthesised substances on the ATP binding site of EGFR, it could be assumed that these compounds might act in the same way. The results of the investigation could be considered as a useful base for future development of potent antimicrobials and antitumor agents among tetrazolo[1,5-c]quinazoline-5-thione S-derivatives.

It is known that tetrazoloquinazolines are capable of tetrazole-azide-azomethine tautomeric transformations due to the electrono-donative influence of the condensed quinazoline ring to the tetrazole one [15]. FT-IR spectral analysis of the synthesized substances (3.1-3.5) in solid form and in chloroform solution showed the absence of the azide group vibration at 2100 сm −1 , which indicates the prevalence of the tetrazole form of the molecule. The low intensity vibrations of the azide tautomer at 2145 сm −1 and its overtone at 2198 сm −1 were detected when trifluoroacetic acid was added to the substances (3.1-3.5) in chloroform solution.
In the 1 Н NMR-spectra, the tetrazolo [1,5-c]quinazoline system of all synthesized substances was characterized according to the proposed structure and available literature data: the one-proton doublet of Н-10 at 8.34-8.55 ppm, Н-7 and Н-9 as the two-proton multiplet at 8.06-7.74, and H-8 as the one-proton triplet at 7.60-7.97 ppm were observed [8]. The signal of SCH 3 was seen in a strong field of spectra at 2.86 ppm as the threeproton singlet for substance 2.1. The proton of the OH group was shown as a wide oneproton singlet at 14.39 ppm for alcohol 4.1 and at 13.54-12.35 ppm for acids 6.1-6.2, and shifted to a stronger field to 11.68 ppm for 4.2. The signal of the SCH 2 -group appeared in a strong field of the spectra as the two-proton triplet at 3.72 ppm for 2.2, at 3.62 ppm for 2.3, and shifted to a low field as the two-proton singlet at 5.22-5.06 ppm for alcohols 4.1, 4.2, аnd to 4.72-3.40 ppm for ethanones 5.1-5.4, and was also observed at 4.85-4.26 ppm for acid 6.1 and acid esters 7.1, 7.3, 7.4, 7.8. The SCH-group was observed as the one-proton triplet at 5.17-5.03 ppm for 7.5-7.7 and as the one-proton singlet at 4.80 ppm for 7.2. The signals of aliphatic substituents of the synthesized substances were registered in a strong field (2.89-0.86 ppm) and aromatic substituent signals were observed in a low field (7.98-7.22 ppm).
In 13 C NMR spectra of substances 7.2 and 7.6, the characteristic signal of the carboxylic group was registered at 170.55 ppm and 170.18 ppm respectively, and the carbonyl group of compound 5.4 at 191.50 ppm. The quinazoline С-5 was detected at 149.32-149.07 ppm, and the peak of the SСН 2 -group was registered at 38.80 ppm for 5.3, and at 49.87-47.84 ppm for 7.2 and 7.6, which definitely confirmed the tetrazolo [1,5-c]quinazoline-5thione formation and S-regioselectivity of alkylation.

Values of BL in acute action test (%)
It is worth mentioning that substances 2.1, 3.2, 3.3, 3.5, 5.4, and 7.3 showed the effect of the hormesis phenomenon (stimulation of activity with a lower concentration) for 0.025 and 0.1 mg/mL doses. Compounds 2.2, 3.1, 7.4, 7.5 possessed moderate inhibition. The highest toxicity (inhibition Bl and growth to 0%) was shown by 4-nitro-2-((tetrazolo [1,5c] Thus, the SAR study revealed that the introduction of the aliphatic acid ester group resulted in the initiation of luminescent bacteria growth in the chronic test. But replacement of the alkyl group with carboxylic acid or the aromatic acid ester, 2-methyl-4-nitrophenol and ethanone substituents in the 5 th position of the 5-thio-tetrazolo [1,5-c]quinazoline led to increased cytotoxic activity against Photobacterium leiognathi Sh1. The high levels of growth inhibition served as a cytotoxicity marker of potential possession of the antitumor activity. 0 DMSO (control) 100.0 74.5 127.7 127.7 a substances 4.1, 6.2, 6.3, 7.2 were not tested.

Antimicrobial and antifungal activities
All of the newly synthesized compounds were evaluated for their in vitro antibacterial activity against Gram positive bacteria (Staphylococcus aureus, Enterococcus faecalis), Gram negative bacteria (Enterobacter aerogenes, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae), and antifungal properties against Candida albicans. The agar-diffusion method was used for the determination of preliminary activity compared to well-known reference antimicrobials. All of the compounds were dissolved in DMSO in a concentration of 100 µg/disk, using inhibition zone diameter (IZD, mm) as a measure for the antimicrobial activity ( Table 3).
The SAR determined that the introduction of the chloropropyl substituent in substance 2.3 was essential for its antifungal activity against Candida albicans like Nystatin action in the same concentration, but wasn't enough to possess antibacterial properties. Replacing the alkyl substituent by ethylaminodialkyl for substances 3.1-3.3 broadened the range of their antimicrobial activity. Also, shortening of the dialkylamino fragment of substances 3.1-3.3 led to a moderate decrease in antimicrobial activity against Enterococcus faecalis, and otherwise led to an increase in antibacterial properties against Staphylococcus aureus and Escherichia coli, and antifungal properties against Candida albicans. Derivatives with ethylaminohetaryl radicals (3.4, 3.5) showed no antimicrobial properties. 2-(Tetrazolo [1,5-c]quinazolin-5-ylthio)ethanol (4.1) had some antimicrobial activity, but the presence of the 4-nitrophenol fragment of substance 4.2 led to the demonstration of light inhibition of Staphylococcus aureus growth. Introduction of the ethanone moiety into the structure of the synthesized compounds (5.1, 5.2, 5.4) had no influence on the mentioned activity. An exception was substance 5.3 with a 4-methoxyphenyl radical at the 5 position of tetrazolo [1,5-c]quinazoline-5-thione, which demonstrated the best results for the antibacterial and antifungal properties among all the synthesized compounds against Staphylococcus aureus, Escherichia coli, and Candida albicans, and was the only one that inhibited growth of Pseudomonas aeruginosa and Klebsiella pneumoniae. The impact of the halogenocarboxylic acid and ester fragments on substances 6.1-6.3 and 7.1-7.8 led to the disappearance of all their antimicrobial activities. Enterococcus faecalis was nonsensitive to all of the synthesized compounds.

Anticancer assay for preliminary in vitro testing
Considering non-small-cell lung, colon cancer, melanoma, and ovarian cancer, they were practically insensitive to the synthesized substances except for the demonstration of the light antitumor activity by 1-(4-methoxyphenyl)-2-(tetrazolo [1,5-c] So, according to the SAR, introduction of the (4-tolyl)ethanone (5.2), acid (6.2, 6.3), and ester (7.1) fragments into the molecule of synthesized compounds imparted them the lethal activity against acute lymphoblastic leukemia CCRF-CEM cell line, among which ethyl 2-(tetrazolo [1,5-c]quinazolin-5-ylthio)acetate (7.1) was the most active. The substitution of the 4-methyl group of substance 5.2 with the 2,5-dimethoxy group for 5.4 changedthe range of its antitumor activity greatly to the widest moderate one among all investigated compounds with the best results inhibiting the CNS cancer SNB-75 cell line. But, unfortunately all of these compounds haven't satisfied the predetermined threshold inhibition criteria to progress to the 5-dose screen, which was designed to efficiently capture compounds with antiproliferate activity.

Docking, scoring, and visual inspection of synthesized substances into the ATP binding site of EGFR
To help us understand the anticancer activity of the observed compounds and guide further SAR studies, molecular docking of compounds 2.1, 2.2, 5.1-5.4, 6.1-6.3, and 7.1 into the ATP binding site of EGFR (2ITY.pdb) was performed [31]. The crystal structure of the enzyme with Gefitinib was obtained from the protein data bank [32]. The selected molecular targets cover some of the basic molecular mechanisms of antitumor effects. Also, the choice of the biological target was due to the literature data on the mechanism of action of 4-R-anilinoquinazolines -known anticancer products [17].
According to Consensus score, compound 6.2 showed the highest affinity for EGFR, but was still lower compared to other known drugs (Gefitinib, Lapatinib). The calculated values are presented in Table 5. Thus, after comparing the docking mode, it could be postulated that the designed compounds might act on the same enzyme target where Gefitinib acted.
Although the results of the antimicrobial activity are not very potent, the anticancer results are quite promising. Compared to the docking mode, it could be assumed that the synthesized substances might act on the ATP binding site of EGFR like Gefitinib does. And they could be used for advanced QSAR analysis based on in vitro biological activity investigations for the further purposeful optimization of the leading compounds in the more effective antimicrobials because of the ever-mounting problem of microorganisms' resistance as well as novel anticancer agents.

Experimental
Melting points were determined in open capillary tubes in a Thiele's apparatus and were uncorrected. The elemental analyses (C, H, N, S) were performed using the ELEMENTAR vario EL cube analyzer. UV-vis spectra (190-400 nm) were recorded on the Analytic Jena UV-vis spectrophotometer Specord 200 in methanol. The IR spectra (4000-600 cm −1 ) were recorded on a Bruker ALPHA FT-IR spectrometer using a module ATR eco ZnSe. 1 H NMR spectra (400 MHz and 500 MHz) were recorded on the Varian-Mercury 400 and Bruker Avance DRX-500 spectrometers with SiMe 4 as the internal standard in DMSO-d 6 solution. The LC-MS was recorded using a chromatography/mass spectrometric system which consisted of the high-performance liquid chromatograph "Agilent 1100 Series" equipped with the diode-matrix and mass-selective detector "Agilent LC/MSD SL" (atmospheric pressure chemical ionization -APCI). The purity of all obtained compounds was checked by 1 H NMR and LC-MS.
Other starting materials and solvents were obtained from commercially available sources and used without additional purification.

Bioluminescence inhibition test
The marine luminescent bacteria Photobacterium leiognathi strain Sh1, isolated from the Azov Sea Shrimp, were used for the bioluminescence analysis. Bacteria were cultivated on a nutrient environment containing (g/L): pepton -5, yeast extract -1.5, meat extract -1.5, sodium chloride -30, pH 7.4. In the acute action test (inhibiting luminescence of bacteria), bacteria were diluted with the 3% sodium chloride solution up to the concentration 10 5 cell/mL. The 5 -50 µg/mL of the studied substances suspended in DMSO were mixed with 1 mL of the diluted bacterial suspension. Vials were incubating for 10 minutes at 25 °C, then the intensity of bioluminescence was measured in % relatively to the control tests which were prepared without the studied compounds. In the chronic action test (inhibiting growth and luminescence of bacteria), growth environment was added to the eventual breeding 1:50 and was incubated for 16-18 hours at 30 °С, whereupon the intensity of bioluminescence was measured as well as in the previous method. Tetracycline was used as a reference. The bacterial luminescence was measured with the Bioluminometer BLM-8801 («Science», Krasnoyarsk, Russia).

Antimicrobial and antifungal test
All the newly synthesized compounds were evaluated for their in vitro antibacterial activity in the Zaporozhye Regional Hospital Bacterial Laboratory against Gram positive bacteria (Staphylococcus aureus ATCC 25923, Enterobacter aerogenes, E. faecalis ATCC 29212), Gram negative bacteria (Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 25922, Klebsiella pneumoniae 68). They were also evaluated for their in vitro antifungal potential against Candida albicans ATCC 885653. The amount of microbial cells was 1.5 * 10 8 c.f.u./mL. The incubation period of bacteria was 24 h at 35 °C, yeast -48-72 h at 28-30°С. The agar-diffusion method was used for the determination of the preliminary antibacterial and antifungal activity. Standard sterilized filter paper discs (6 mm diameter) impregnated with a solution of the test compound in DMSO (100 µg/disk) were placed on an agar (Müller-Hinton Broth (Oxoid)) plate seeded with the appropriate test organism in triplicates. DMSO alone was used as the control at the same above-mentioned concentration. Ampicillin, Ceftazide, Amikacin, Gentamycin, Ceftriaxone, and Nystatin were used as reference drugs. The results were recorded for each tested compound as the average diameter of inhibition zones diameters (IZD) of bacterial or fungal growth around the discs in mm.

Antitumor activity
The primary anticancer assay was performed against the human tumor cell lines panel derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda [28][29][30]. The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells were inoculated in 96 well microtiter plates in 100 mL assay volume, at plating densities ranging from 5000 to 40000 cell/well. After cell inoculation, the microtiter plates were incubated at 37°C under an atmosphere of 5:95 CO 2 :air (v/v) at 100% relative humidity, for 24 h prior to the addition of drugs under assessment. Following drug addition (1 µM), the plates were incubated for an additional 48 h, under the same conditions. Sulforhodamine B (SRB) solution (100 µL, 0-4% w/v in 1% aq. acetic acid) was added to each well and plates were incubated for 10 min at room temperature. The percentage growth was evaluated spectrophotometrically versus the controls not treated with test agents.

Docking, scoring, and visual inspection of synthesized substances into ATP binding site of EGFR
Research was conducted by flexible molecular docking, as an approach for finding molecules with affinity for a specific biological target using the software package OpenEye, including related utilities: Fred Receptor2.2.5, Vida4. 1.1,Flipper,Babel3,Omega2.4.3,and Fred2.2.5 [33]. The crystal structure of the enzyme EGFR (2ITY.pdb) with Gefitinib was obtained from the protein data bank [32].
The methodology of research consisted of the following steps: o generation of R-, S-and cis-, trans-isomers of ligands (the studied compounds and relevant drugs, program Flipper), which allowed the production isomer's range of studied compounds; o generation of 3D-structure of the obtained isomeric forms -molecular modelling (Hyper Chem 7.5) using the method of molecular mechanics (MM +) and semiempirical quantum mechanical method with Polak-Ribiere algorithm (PM3); o generation of conformations of ligands (Omega2.4.3). The number of conformations obtained wasn't significant due to the further selection by program Fred2.2.5 most optimal conformer; o carrying out molecular docking (Fred2.2.5).
A number of scoring functions (Shapegauss, PLP, Chemgauss2, Chemgauss3, Chemscore, OEChemscore, Screenscore, CGO, CGT, Zapbind, Consensus Score) was obtained as a result of the studies, values of which assess specific characteristics of the ligand-protein complex, indicating the possibility of their matching.