Synthesis and Anticancer Activity of 2-(Alkyl-, Alkaryl-, Aryl-, Hetaryl-)-[1,2,4]triazolo[1,5-c]quinazolines

The combinatorial library of novel potential anticancer agents, namely, 2-(alkyl-, alkaryl-, aryl-, hetaryl-)[1,2,4]triazolo[1,5-c]quinazolines, was synthesized by the heterocyclization of the alkyl-, alkaryl-, aryl-, hetarylcarboxylic acid (3H-quinazoline-4-ylidene)hydrazides by oxidative heterocyclization of the 4-(arylidenehydrazino)quinazolines using bromine, and by the heterocyclization of N-(2-cyanophenyl)formimidic acid ethyl ester. The optimal method for synthesis of the s-triazolo[1,5-c]quinazolines appeared to be cyclocondensation of the corresponding carboxylic acid (3H-quinazoline-4-ylidene)hydrazides. The compounds’ structures were established by 1H, 13C NMR, LC- and EI-MS analysis. The in vitro screening of anticancer activity determined the most active compound to be 3,4,5-trimethoxy-N′-[quinazolin-4(3H)-ylidene]benzohydrazide (3.20) in micromolar concentrations with the GI50 level (MG_MID, GI50 is 2.29). Thus, the cancer cell lines whose growth is greatly inhibited by compound 3.20 are: non-small cell lung cancer (NCI-H522, GI50=0.34), CNS (SF-295, GI50=0.95), ovarian (OVCAR-3, GI50=0.33), prostate (PC-3, GI50=0.56), and breast cancer (MCF7, GI50=0.52), leukemia (K-562, GI50=0.41; SR, GI50=0.29), and melanoma (MDA-MB-435, GI50=0.31; SK-MEL-5, GI50=0.74; UACC-62, GI50=0.32). SAR-analysis is also discussed.

In the 1 H NMR spectra of compounds 4.1-4.6, there was a characteristic singlet of the azomethine proton at 8.80-8.30 ppm and a low-field broadened NH singlet of the quinazoline cycle at 11.20-15.60 ppm. In addition, the synthesized compounds were also characterized by the ABCD-system of the quinazoline cycle and substituents with appropriate multiplicity. The location of the azomethine group's proton signals at 8.80-8.30 ppm provides an opportunity to affirm the trans arrangement of substituents around the double bond of the azomethine group [19]. Annelation of the triazole ring to the quinazoline cycle was also performed by heterocyclization of the alkyl (arylalkyl-, aryl-) carboxylic acid (3H-quinazolin-4-ylidene)hydrazides (3.1-3.40) in glacial acetic acid or by oxidative heterocyclization of 4-arylidenehydrazinoquinazolines (4.1-4.6) with bromine in glacial acetic acid (Scheme 1) [21]. In addition, compounds 5.7, 5.12, 5.17, 5.18, 5.23, 5.27, 5.31, 5.33 were obtained by the heterocyclization of N- (2-cyanophenyl)formimidic acid ethyl ester with aromatic acid hydrazides (Scheme 1). The latter method was less effective than the previous because of a lower yield. It is important, that in all cases, intermediate [1,2,4]triazolo [4,3-c]quinazolines are ANRORC-rearranged forming 2-R- [1,2,4]triazolo [1,5-c]quinazolines (5.1-5.40) [21]. The 1 H-NMR spectra of compounds 5.1-5.40 were significantly different from the spectra of hydrazides 3.1-3.40 and hydrazones 4.1-4.6. Thus, the characteristic low-field singlet H-5 of the s-triazolo [1,5-c]quinazolines ring resonated at 9.85-9.25 ppm. It is important that the chemical shift of this proton was associated with the donor-acceptor properties of the substituent at position 2. In the case of pronounced donor properties of the substituents (5.1-5.4, 5.7, 5.11, 5.14, 5.20), the H-5 singlet was shown at 9.36-9.25 ppm, while the acceptor substituents displaced it in the low-field part of the spectrum (9.85-9.60 ppm). Other protons of the quinazoline cycle were registered at 8.56-7.71 ppm as consecutive doublets (H-10 and H-7) and triplets (H-8 and H-9). In addition, compounds 5.1-5.40 were characterized by corresponding multiplicity and chemical shifts of the functional groups at position 2 of the protons signals.
The signals of carbon at the 2 аnd 5 positions in the 13 C NMR spectra were characteristic for triazolo [1,5-c]quinazolines (compounds 5.2, 5.4, 5.12, 5.17, 5.24, 5.32, 5.34, 5.37, 5.38). The carbon in position 2 was electron-deficient and registered in the low-field at 159.47-164.40 ppm. At the same time, the carbon in position 5, even though it is located between two nitrogen atoms, was less deshielded and was found at 134.32-139.69 ppm.
In the LC-MS spectra, compounds 5.1-5.40 were characterized by positive ions [M+1]. The spectra of compounds 3.23-3.27 with chlorine atoms in the molecule have additional molecular ion [M+3]. The mass spectra (EI) of the 2-R- [1,2,4]triazolo [1,5-c]quinazolines (5.1, 5.2, 5.7, 5.9, 5.12, 5.14, 5.15, 5.18, 5.19, 5.22, 5.26, 5.27, 5.30-5.38) showed the aromaticity of the tricyclic system: the molecular peak M +• for the majority of compounds had the maximum intensity. The molecular peak of these compounds was characterized by fragmentation of the main line of the C(10b)-N(1) and N(3)-N(4) with cleavage of the amidine fragment ([RC(N)N] + ) and by the formation of the ion whose mass corresponded to the calculated quinazoline mass (m/z 129). In the latter case, the destruction of the triazole cycle occurred. The further expansion pattern was typical for the quinazoliniumcation.
Some of the synthesized compounds had peculiarities of fragmentation under electron impact. Thus, the spectrum of compound 5.1 had two equivalent directions of [M] +• fragmentation, firstly, by the N(1)-C (2) and N(3)-N(4) bonds with the formation of the ion [M-C 2 H 3 N] + with m/z 143, and secondly, classically, by the C(10b)-N(1) and N(3)-N(4) bonds with the formation of [CH 3 C(N)N] + and the quinazolinium-cation (m/z 129). This fact indicates that the methyl group in the position 2 destabilized the heterocyclic aromaticity.
The 1 H-NMR spectra of compounds 7.1 and 7.2 were characterized by the low-field singlet H-5, which resonated at 9.52 and 9.25 ppm respectively, protons of the quinazoline cycle at 8.54-7.71 ppm were found as consecutive doublets (H-10 and H-7), and triplets (H-8 and H-9). The 13 C NMR spectrum of compound 7.1 was characterized by the signals of the carbon atoms in position 2 and 5 at 159.92 ppm and 139.69 ppm. The sp 2 hybridized carbon atom of the CO-group of 7.1 was observed in the low-fields at 156.05 ppm. The mass spectrum (EI) of [1,2,4]triazolo [1,5-c]quinazoline-2-ylpropionic acid (7.2) was characterized by a low intensity peak of the molecular ion M +• , due to the presence of a carboxyl group. The primary process of the 7.2 M +• fragmentation was specified by the elimination of the OH and COOH radicals ([M-OH] +• is 9.5% and [M-COOH] + is 100%). Further decay patterns coincided with the previously given 2-R- [1,2,4]triazolo [1,5-c]quinazolines destruction direction, i.e. there was destruction of the 1,2,4-triazole ring by the C(10b)-N(1) and N(3)-N(4) bonds, followed by fragmentation of the quinazoline bicycle.
In the 1 H-NMR spectra of compound 7.3, a two-proton multiplet was observed at 8.39 ppm, which characterized the proton at position 2 and 5 of the quinazoline cycle. Other aromatic protons of compound 7.3 formed a broad multiplet (8.20-7.60 ppm) with an intensity of seven proton units.
The 13 C NMR spectrum of compound 7.3 was characterized by the equivalent signals of the unshielded carbon atoms in position 1 and 3 of the izoindol cycle at 166.18 ppm. The mass spectrum (EI) of the latter compound was characterized by the molecular ion (M +• , m/z 290, 49.5%), whose main ways of destruction were related to the elimination of particles CO and OH and formed the fragmented ion with m/z 245 (100%).

Through dose-dependent study in vitro on 60 cancer cell lines
Compounds 3.20 and 5.10 were chosen by the NCI for dose-dependent action in five concentrations according to a standard procedure in 58 cell lines of nine types of cancer (100μM-0.01μM), and was investigated [21][22][23]. Three dose-dependent parameters were calculated: 1) GI 50 -molar concentration of the compound that inhibits 50% of net cell growth; 2) TGI -molar concentration of the compound leading to the total inhibition of cell growth; 3) LC 50 -molar concentration of the compound leading to 50% net cell death. Furthermore, the mean graph midpoints (MG_MID) were calculated for each of the parameters, giving an average activity parameter over all of the cell lines for tested compounds. For the calculation of the MG_MID, insensitive cell lines are included with the highest tested concentration ( Table 2).

General procedure for (3Н-quinazolin-4-ylidene)carbohydrazides (3.1-3.41)
Method A. To a solution of 1.65 g (10 mM) of 4-chloroquinazoline (1.1) in 10 ml of dioxane, the corresponding carboxylic acid hydrazide (11 mM) was added and kept in a water bath at 60°C for 8 h. After cooling, the reaction mixture was poured into the water, and a 5% solution of sodium bicarbonate was added to achieve pH 6-7. The formed precipitate was filtered off and dried.

Method B.
To a solution of the corresponding carboxylic acid (11 mM) in 10 ml of anhydrous dioxane, 1.95 g (11 mM) of carbonyldiimidazole was added and heated in a water bath at 60-80°C for 1 hour, under the calcium chloride tube. While stirring, 4-hydrazinoquinazoline (1.1) was added to the reaction mixture 1.60 g (10 mM) and left for 8 h at room temperature. The mixture was poured into the water and acetic acid was added to achieve pH 6-7. The formed precipitate was filtered off and dried.
Method B. (5.12, 5.17, 5.23, 5.25, 5.27) To a solution of 2 mM of N-(R-benzyliden)-N′-(3Hquinazolin-4-ylidene)hydrazine (4.1-4.6) in 20 ml of glacial acetic acid, 0.5 g (6 mM) of sodium acetate was added with stirring at room temperature. Then a solution of 0.32 g of bromine (2 mM) in 10 ml of glacial acetic acid was added dropwise to a mixture. The mixture was stirred for 1 hour, then was poured into crushed ice (100.0 g). The formed precipitate was filtered off and dried.
Method C. (5.7, 5.12, 5.17, 5.18, 5.23, 5.23, 5.27) 1.18 g (10 mM) of o-aminobenzonitrile (1.1a) in 5 ml of triethyl orthoformate was refluxed for 3 h, and after this the mixture was distilled off. To the resulting mixture, 10 mM of the corresponding carboxylic acid hydrazides and 5 ml of glacial acetic acid were added and refluxed for 4 h. The solvent was distilled off and methanol was added. The mixture was stirred for 30 min. The formed precipitates were filtered off and dried.

Cytotoxic activity against malignant human tumor cells
The primary anticancer assay was performed against a human tumor cell lines panel derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda [21][22][23]. 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. End point determinations were made with the protein binding dye, 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 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 concentration levels. Percentage growth inhibition was calculated as: [(T i − T z )/(C − T z )] × 100 for concentrations for which T i ≥ T z , [(T i − T z )]/T z ] × 100 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 with the most sensitive cell lines.