Synthesis of New 6-{[ω-(Dialkylamino(heterocyclyl)alkyl]thio}-3-R-2H-[1,2,4]triazino[2,3-c]quinazoline-2-ones and Evaluation of their Anticancer and Antimicrobial Activities

Several novel 6-thio-3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-based compounds containing an ω-(dialkylamino(heterocyclyl)]alkyl fragment were synthesized to examine their anticancer activity. Some of the 6-{[ω-(hetero-cyclyl)alkyl]thio}-3-R-2H-[1,2,4]triazino[2,3-c]quinazoline-2-ones (3.1–3.10) were obtained by the nucleophilic substitution of 6-[ω-halogenalkyl]thio-3-R-2H-[1,2,4]triazino[2,3-c]quinazoline-2-ones (2.1–2.8) with azaheterocycles. Alternatively, compounds 3.1–3.22 were synthesized by alkylation of 3-R-6-thio-2H-[1,2,4]triazino[2,3-c]quinazoline-2-ones potassium salts (1.1–1.4) with (2-chloroethyl)-N,N-dialkylamine hydrochlorides or 1-(2-chloroethyl)heterocycle hydrochlorides. The structures of compounds were elucidated by 1H, 13C NMR, LC–MS and EI-MS analysis. Then anticancer and antibacterial, bioluminescence inhibition of Photobacterium leiognathi Sh1 activities of the substances were tested in vitro. It was found that compound 3.18 possessed a wide range of anticancer activity against 27 cell lines of cancer: non-small cell lung, colon, CNS, ovarian, renal, prostate, breast, melanoma and leukemia (log GI50 < −5.65). The “structure-activity” relationship was discussed. COMPARE analysis for synthesized anticancer active compounds was performed.


Introduction
The quinazoline skeleton is a heterocyclic system that can be found in many prospective anticancer drugs [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. Some derivatives, notably 4-R-phenylaminoquinazolines (drugs «Iressa» (I), «Erlotinib» (II), «Vandetanib» (III) and others) are ATP-competitive irreversible inhibitors of the tyrosine-kinase epidermal growth factor receptor and others protein-kinase enzymes, which are widely used in oncological practice (Scheme 1) [3,5]. It's important that anticancer activity of 4-R-phenylaminoquinazolines is determined by both the base heterocycle and aniline fragment in 4 th position of molecule and requires the presence of halogens, hydroxy and cyano group. To improve the pharmacokinetic properties, notably bioavailability and lipophilicity, it is advisable to introduce appropriate functional groups in 6 th and 7 th position of molecule. Such anintroduction of acryl-or butyn-2-amide fragments leads to the increase of metabolism and elimination and decrease of accumulation, while methoxy-and heterocyclylalkyloxy-groups are necessary for the improvement of hydrophobic interaction with the appropriate enzymes.

Sch. 2.
Structures of quinazoline-based compounds and their medicinal chemistry optimization.

Tab. 2.
Antimicrobial activity of synthesized compounds

Antimicrobial and antifungal activities
The results of antimicrobial screening showed that researched substances had significant antimicrobial activity only against cereous bacteria Mycobacterium luteum (Table 2). Thus, the highest antibacterial data were established for 6-[(2-dialkylaminoethyl)thio]-3-R-2H- [1,2,4]triazino [2,3-c] position, but elongation of radical up to propyl or butyl leads to its significant reduction; 3) antimicrobial activity against M. luteum is characteristic for the majority of compounds, and compounds with phenyl, thionyl or р-methoxyphenyl substituents at 3rd position have the strongest activity.

3.11-3.22)
had the highest antimicrobial activity against M. luteum, testifying the potential presence of antituberculosis activity of the mentioned compounds.

Anticancer assay for preliminary in vitro testing
Newly synthesized compounds were selected by the National Cancer Institute (NCI) Developmental Therapeutic Program for the in vitro cell line screening to investigate their anticancer activity. Compounds 3.1, 3.14-3.16, 3.18, 3.21 were submitted and evaluated according to the US NCI protocol [23][24][25][26][27][28]. The compounds were first evaluated at one dose primary anticancer assay toward or approximately 60 cell lines (concentration 10 −5 M). The human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate and breast cancers. In the screening protocol, each cell line was inoculated and preincubated for 24-48 h on a microtiter plate. Test agents were then added at a single concentration and the culture was incubated for an additional 48 h. End point determinations were made with a protein binding dye, sulforhodamine B (SRB). Results for each test agent were reported as the percent growth of the treated cells when compared to the untreated control cells. The preliminary screening results are shown in Table 3.
Investigation of the compounds 3.1, 3.14-3.16, 3.18, 3.21 showed that individual cell lines had different sensitivity towards synthesized compounds in concentration 10 −5 М (Table 3) The dose-dependent action in 5 concentrations according to standard procedure of NCI (100μM-0.01μM) was researched for 3.14, 3.16, 3.18. The 3 dose-dependent parameters were calculated: 1) GI 50 -molar concentration of the compound that inhibits 50% net cell growth; 2) TGI -molar concentration of the compound leading to total inhibition of cell growth; 3) LC 50 -molar concentration of the compound leading to 50% net cell death. If logarithmic data of researched parameters (log GI 50 , log TGI та log LC 50 ) was less than −4.00, substances were marked as active. For each of the parameters the average experimental data were calculated (mean graph midpoints, MG_MID) ( Table 4).

Tab. 4.
Summary of anticancer screening data at dose-dependent assay It is important to note that compound 3.14 had the highest anticancer activity against cell

COMPARE analysis and molecular mechanism assumptions
We have also performed COMPARE analyses for all the active compounds to investigate the similarity of their cytotoxicity (mean graph fingerprints) with those of known anticancer standard agents, NCI active synthetic compounds and natural extracts, which are present in public available databases [29][30][31][32]. Such analysis is based on comparing the patterns of differential growth inhibition for cultured cell lines and can potentially gain insight into the mechanism of the cytotoxic action. It determines Pearson correlation coefficient (PCC) for the degree of similarity of mean graph fingerprints obtained from in vitro anticancer screen with patterns of activity of standard agents. We performed COMPARE computations for synthesized compounds against the NCI 'Standard Agents' database at the GI 50 level (correlations PCC >0.4) ( Table 6).
COMPARE analysis hypothesis precludes that the compounds 3.14, 3.16 and 3.18 might have the same mechanism of action as the agent with known action mechanism, if the data pattern of a compound correlates well with the data pattern of compounds belonging to the standard agent database. The majority of significant correlations for 3-R-6-thio-2H- [1,2,4]triazino [2,3-c]quinazoline-2-ones derivatives were found with inhibitor of topoisomerases I and II, as well as inhibition or promotion of microtubules polymerization and CTP-synthase inhibitor. These molecular targets should be considered as the first priority and be explored for the leukemia, colon, CNS, renal cancers cell lines.

General methods
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. IR spectra (4000-600 cm -1 ) were recorded on a Bruker ALPHA FT-IR spectrometer (Bruker Bioscience, Germany) using a module for measuring attenuated total reflection (ATR). 1 H NMR spectra (500 MHz) and 13 C NMR spectra (100 MHz): were recorded on a Varian-Mercury 400 (Varian Inc., Palo Alto, CA, USA) spectrometers with TMS as internal standard in DMSO-d 6 solution. LC-MS were recorded using chromatography / mass spectrometric system which consists of high performance liquid chromatograph «Agilent 1100 Series» (Agilent, Palo Alto, CA, USA) equipped with diode-matrix and mass-selective detector «Agilent LC/MSD SL» (atmospheric pressure chemical ionization -APCI). Electron impact mass spectra (EI-MS) were recorded on a Varian 1200 L instrument at 70 eV (Varian, USA). The purity of all obtained compounds was checked by 1 H-NMR and LC-MS.  [18,21]. Other starting materials and solvents were obtained from commercially available sources and used without additional purification.

Antimicrobial and antifungal test
The investigation of antimicrobial and antifungal activity of compounds 2.1-2.8 and 3.1-3.22 was carried out with the stiff plate agar diffusion method against Escherichia coli, Staphylococcus aureus, Mycobacterium luteum, Candida tenuis and Aspergillus niger. The amount of microbial cells was 109 c.f.u./mL. Incubation period of bacteria was 24 h at 35°C, yeast -48 to 72 h at 28-30°C. Antibiotics vancomicin, oxacillin, nystatin were used as standards. The bacterial cultures, standards and the obtained substances were streaked across grooves at 5 mg/mL concentration, and then allowed to diffuse in the agar nutrient plate. The antimicrobial effect and degree of activity of the tested compounds were evaluated by measuring of the inhibition zone diameters (low sensitive: 11-15 mm; sensitive: 16-25 mm; highly sensitive >25 mm). All experiments were repeated three times.

Cytotoxic activity against malignant human tumor cells
Primary anticancer assay was performed at human tumor cell lines panel derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda [24][25][26][27][28][29]. Tested compounds were added to the culture at a single concentration (10 −5 M) and the cultures were incubated for 48 h. End point determinations were made with a protein binding dye, sulforhodamine B (SRB). Results for each tested compound were reported as the percent of growth of the treated cells when compared to the untreated control cells. The percentage growth was evaluated spectrophotometrically versus controls not treated with test agents. The cytotoxic and/or growth inhibitory effects of the most active selected compounds were tested in vitro against the full panel of about 60 human tumor cell lines at 10-fold dilutions of five concentrations ranging from 10 −4 to 10 −8 M. A 48-h continuous drug exposure protocol was followed and an SRB protein assay was used to estimate cell viability or growth. Using the seven absorbance measurements [time zero, (T z ), control growth in the absence of drug (C), and test growth in the presence of drug at the five concentration levels (T i )], the percentage growth was calculated at each of the drug concentrations levels. Percentage growth inhibition was calculated as: for concentrations for which T i ≥ T z , for concentrations for which T i < T z .
Three dose response parameters were calculated for each compound. Growth inhibition of 50% (GI 50 ) was calculated from [(T i − T z )/(C − T z )] × 100 = 50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB staining) as compared to the net protein increase seen in the control cells. The drug concentration resulting in total growth inhibition (TGI) was calculated from T i = T z . The LC 50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment was calculated from [(T i − T z )/T z ] ×100 = -50. Values were calculated for each of these three parameters if the level of activity was reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested. The log GI 50 , log TGI, log LC 50 were then determined, defined as the mean of the log's of the individual GI 50 , TGI, LC 50 values. The lowest values were obtained