Synthesis of 6-N-R-Tetrazolo[1,5-c]quinazolin-5(6H)-ones and Their Anticancer Activity

Chemical compounds with tetrazole ring are very interesting systems that can be valuable in pharmaceutical and clinical applications, especially as anticancer agents. In this work, novel 6-N-R-tetrazolo[1,5-c]quinazolin-5(6H)-ones were synthesized. A large set of IR, LC-, EI-MS, H, C NMR and elemental analysis data were collected and evaluated for their structures and purity. Details of synthesis, namely the N-alkylation, are discussed, including reactions with secondary and tertiary amides. Four new synthesized compounds (2.7, 3.2, 5.2, 5.3) were tested in vitro for anticancer activity at 10 μM against 60 cell lines of nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers. Further synthesis of substances within the series of substituted tetrazolo[1,5-c]quinazoline systems will be attempted to develop improved compounds with better anticancer activity.


1. 1. General Methods
Melting points were determined in open capillary tubes in a «Stuart SMP30» apparatus and are uncorrected. The elemental analyses (C, H, N) were performed using the ELEMENTAR vario EL cube analyzer. 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 13 C NMR spectra (100 MHz) were recorded at a Varian-Mercury 400 and Bruker Avance DRX-500 spectrometers with SiMe 4 as internal standard in DMSO-d 6 solution. LC-MS were recorded using chromatography/mass spectrometric system which consists of high-performed liquid chromatograph «Agilent 1100 Series» 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.

1. Anticancer Assay for Preliminary in vitro Testing
From the newly synthesized compounds 4 substances, namely 2.7, 3.2, 5.2, 5.3 were selected by the NCI Developmental Therapeutic Program for in vitro cell line screening to investigate their anticancer activity. The human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate and breast cancers. Initially, a single high concentration was used (10 μM) in the full NCI 60cell panel. In the screening protocol, each cell line was inoculated and preincubated for 24-48 h on a microtiter plate. Then test substances were added to the plate and the culture was incubated for further 48 h. End point determinations were made with a protein binding dye, sulforhodamine B. Results for each test agent were reported as the percent growth of the treated cells when compared to the untreated control cells (Table 1).
To find the most efficient way of synthesis, various reaction conditions were explored. At first, tetrazolo [1,5c]quinazolin-5(6H)-one (1.1) was dissolved in DMF with equimolar amount of sodium hydride. The corresponding halogen derivative was added only after all hydrogen has been released. The resulting mixture was refluxed for 2 h. Alternatively, the reaction was performed by the addition of potassium carbonate in DMF or sodium bicarbonate in dioxane. The best yields and purity of derivates 2 were observed in the presence of sodium hydride. This method was chosen as the primary one. The next step was the synthesis of acetamides. Firstly 2-(5-oxotetrazolo [1,5-c]quinazolin-6(5H)-yl)acetic acid should be obtained with further aminolysis. The direct alkylation of tetrazolo[1,5-c]quinazolin-5(6H)-one (1.1) with chloroacetic acid has not resulted in the desirable product. Thus, an alkaline hydrolysis of 2-(5-oxotetrazolo[1,5-c]quinazolin-6(5H)-yl)acetate esters 3.1-3.3 was necessary (Scheme 1). However, the cleavage of quinazoline cycle was observed, and the product of the cleavage turned out to be 2-((2-(1H-tetrazol-5-yl)phenyl)amino)acetic acid (4.1) (see further discussion on spectral data).
Then, N-alkylation of tetrazolo[1,5-c]quinazolin-5(6H)-one (1.1) with chloroacetamides was used to synthesize amides 5.1-5.4. The reaction was quite interesting, since the products obtained were disubstituted compounds 5.1-5.4. In this reaction NH proton of acetamide was acting as a competitive acid moiety, which results in the alkylation of the quinazolin-5(6H)-one NH group, and of acetamide NH group of intermediate alkylated product (Scheme 2). This was confirmed by LC-MS and 1 H NMR spectra of the synthesized compounds with intensive peaks of molecular ions with a mass of two acetamide residues. Alkylation with tertiary amides 6.1-6.4 has not revealed any unexpected products (Scheme 2).
The identity of the synthesized compounds was confirmed by IR, LC-, EI-MS, 1 H, 13 C NMR, and elemental analysis. LC-MS of the synthesized compounds in a »soft« ionization (chemical ionization at atmospheric pressure) allowed to register the molecular ion peak [M+1] in high intensity. For compound 4.1 the ion with molecular weight of 220 was observed, confirming cleavage of the quinazoline ring.
In the 1 H NMR spectra of tetrazolo[1,5-c]quinazolin-5(6H)-ones the clear splitting of aromatic quinazoline protons' signals was observed. Thus, H-10 signal can be found at a range of 8.19-8.46 ppm, H-9 at 7.71-7.95 ppm, H-7 at 7.50-7.85 ppm, and H-8 at 7.42-7.61 ppm. The signals for these protons for some compounds were overlapping with each other (2.3, 2.7) or with other aromatic substituents protons (5.4). At the same time, for compound 4.1 the diamagnetic shift of aromatic protons was observed, obviously due to the absence of electron-deficient tetrazoloquinazoline system. Thus, Ph-3 signal was registered at 7.80 ppm as a doublet, Ph-4 at 7.30 ppm as a triplet, Ph-5 at 6.72 ppm as a triplet, and Ph-6 as a doublet at 6.66 ppm. Besides that, the NH tetrazole proton was detected as a broad singlet at 8.22-7.93 ppm.
The signal of the NCH 2 group can be used as a confirmation of N-alkylation. For compounds 2.1-2.7 it was registered as a two-proton singlet at 5.54-3.80 ppm, except for compound 2.4, where it was as a two-proton triplet at 4.55 ppm. Due to electron acceptor influence of 2oxo-2-phenylethyl and 2-oxo-2-(p-tolyl)ethyl moiety the signal of NCH 2 group was observed in the weak field at 6.04-6.01 ppm for substances 2.6 and 2.7.  (Table 1).

Conclusions
Due to their unique characteristics, compounds with tetrazole ring have been used in pharmaceutical and clinical applications especially as anticancer agents. In this work, novel 6-N-R-tetrazolo[1,5-c]quinazolin-5(6H)-ones were synthesized. N-Alkylation reaction of tetrazolo [1,5c]quinazolin-5(6H)-one (1.1) with various chloro-derivatives was performed under various reaction conditions. As the best option a reflux in DMF with an equimolar amount of sodium hydride was selected. Spectral data confirm molecular structures of investigated compounds. These investigations will be continued for other activities and core structures.